Alpha-amylase variants having an elevated solvent stability, method for the production thereof and detergents and cleansers containing these alpha-amylase variants

ABSTRACT

The present invention relates to α-amylase variants that are stabilized to solvent-exerted hydrolysis, in particular at elevated temperatures or high pH, by point mutagenesis of asparagine (N) or glutamine (Q) residues located on the surface of the molecule to give other amino acid residues. The invention further relates to methods of increasing the stability of an α-amylase to solvent-exerted hydrolysis, in particular at elevated temperatures or high pH, whereby at least one asparagine (N) or glutamine (Q) residue on the surface of the molecule is replaced with a different amino acid residue. The α-amylase variants obtained thereby exhibit better stability to influences of the solvent, increased processivity, and are suited for numerous industrial areas of use, in particular as active ingredients in detergents and cleansers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a § 365 continuation application of PCT/EP2005/010261 filed Sep.22, 2005, which claims the priority of German patent application DE 102004 047 777.9, filed Oct. 1, 2004, each of the foregoing applicationsis incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to α-amylase variants which have beenstabilized to solvent-exerted hydrolysis, in particular at elevatedtemperatures or high pH, by means of point mutagenesis of surface aminoacids, to methods of preparation thereof via mutagenesis of particularsurface amino acid residues and to detergents and cleansers containingthese α-amylase variants.

BACKGROUND OF THE INVENTION

α-Amylases (E.C. 3.2.1.1) hydrolyze internal α-1,4-glycosidic bonds ofstarch and starch-like polymers with the formation of dextrins andβ-1,6-branched oligosaccharides. They are very much among theindustrially most important enzymes. Thus, for example, α-amylases areemployed in the production of glucose syrup, for the treatment of rawmaterials in the manufacture of textiles, for the production ofadhesives or for the production of sugar-containing food or foodingredients. Another important field of use is the use as an activecomponent in detergents and cleansers.

Since detergents and cleansers have mainly alkaline pH values,particular use is made here of α-amylases that are active in alkalinemedium. These types of α-amylases are produced and secreted bymicroorganisms, i.e. fungi or bacteria, especially those of the generaAspergillus and Bacillus. Starting from these natural enzymes, there isnow quite a vast abundance of variants available which have been derivedvia mutagenesis and have specific advantages depending on the field ofuse.

Examples thereof are the α-amylases of Bacillus licheniformis, B.amyloliquefaciens and B. stearothermophilus and their improveddevelopments for the use in detergents and cleansers. The B.licheniformis enzyme is available from Novozymes under the nameTermamyl® and from Genencor under the name Purastar® ST. Products offurther development of this α-amylase can be obtained from Novozymesunder the trade names Duramyl® and Termamyl® ultra, from Genencor underthe name Purastar® OxAm and from Daiwa Seiko Inc., Tokyo, Japan, asKeistase®. The B. amyloliquefaciens α-amylase is sold by Novozymes underthe name BAN®, and variants derived from the B. stearothermophilusα-amylase are sold under the names BSG® and Novamyl®, likewise byNovozymes.

Examples of α-amylases from other organisms are further developments ofthe Aspergillus niger and A. oryzae α-amylases, obtainable under thetrade name Fungamyl® from Novozymes. Another commercial product isAmylase-LT®, for example.

Mention may further be made of the Bacillus sp. A 7-7 (DSM 12368)α-amylase disclosed in the application WO 02/10356 A2 and of the B.agaradherens (DSM 9948) cyclodextrin glucanotransferase (CGTase)described in the application WO 02/44350 A2. In addition, for example,the applications WO 03/002711 A2 and WO 03/054177 A2 define sequencespaces of α-amylases, all of which could be suitable in principle forcorresponding applications.

The application DE 10309803.8, which has not been prepublished, forexample, describes point mutations for improving the activity of saidenzymes in alkaline medium. According to this application, amino acidsubstitutions suitable here are those in positions 13, 32, 194, 203,230, 297, 356, 406, 414 and/or 474, according to the numbering of theunprocessed B. amyloliquefaciens α-amylase.—These positions areaccording to the numbering of the unprocessed Bacillus sp. A 7-7 (DSM12368) α-amylase (WO 02/10356 A2) L13, T36, W198, S201, 1208, A235,D302, D361, H408, K416 and N476, respectively, with the following,particularly effective substitutions: 13P, 32A, 194R, 197P, 203L, 230V,297D, 356D, 406R, 414S and 474Q.

Another example of point mutagenesis on α-amylases is the application WO00/22103 A1 which discloses polypeptides, inter alia also α-amylasevariants, containing mutagenized surface amino acids. The purpose ofthis mutagenesis was to reduce the immunogenicity and/or allergenicitycaused by these molecules.

Fusion products of α-amylases for the use in detergents and cleansershave also been described. Thus, for example, the application WO 96/23874A1 discloses hybrids of the α-amylases of Bacillus licheniformis, B.amyloliquefaciens and B. stearothermophilus. According to the teachingof this application, such hybrid amylases may be prepared fordetermining the three-dimensional structure of said amylases, in orderto use said structure for detecting important positions for enzymicactivity. Further developments in this respect are the applications WO97/41213 A1 and WO 00/60059 A2, which report numerous α-amylase variantswhose respective performances have been improved. The application WO03/014358 A2 discloses special hybrid amylases of B. licheniformis andB. amyloliquefaciens.

The three applications WO 96/23873 A1, WO 00/60060 A2 and WO 01/66712A2, which are the basis of the commercial product Stainzyme® fromNovozymes, constitute another important prior art. All the variantsobtainable by point mutagenesis which are specified in each of theseapplications have altered enzymic properties and are therefore claimedor described for the use in detergents and cleansers. WO 96/23873 A1makes mention of, in some cases two or more, point mutations in morethan 30 different positions in four different wild type amylases. Theyapparently have altered enzymic properties with regard to thermalstability, oxidation stability and calcium dependence. They includepoint mutations in the following positions, each of which is stated withrespect to the Bacillus sp. NCIB 12512 α-amylase: substitution ofoxidizable amino acids in positions M9, M10, M105, M202, M208, M261,M309, M382, M430 or M440, preferably M91L; M10L; M105L; M202L, T, F, I,V; M208L; M261L; M309L; M382L; M430L and M440L; deletions of F180, R181,G182, T183, G184 and/or K185; and additionally the substitutions K269R;P260E; R124P; M105F, I, L, V; M208F, W, Y; L217I; V206I, L, F; Y243F;K108R; K179R; K239R; K242R; K269R; D163N; D188N; D192N; D199N; D205N;D207N; D209N; E190Q; E194Q or N106D.

The application WO 00/60060 A2 likewise specifies a multiplicity ofpossible amino acid substitutions in 10 different positions, on thebasis of two very similar α-amylases from two different microorganisms,with the same numbering α-amylases AA349 and AA4560). These are thefollowing sequence variations: R181*, G182*, D183*, G184*;

N195A, R, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;

1206A, R, D, N, C, E, Q, G, H, L, K, M, F, P, S, T, W, Y, V;

E212A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;

E216A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V;

K269A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V; and/or

R181A, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V. Thisdevelopment too is against the background of improving performance viamutations.

WO 01/66712 A2, finally, refers to 31 different amino acid positionswhich are partially identical to the ones mentioned above and which havebeen mutated in either of the two α-amylases specified in theapplication WO 00/60060 A2 and which are said to improve aspects of bothperformance and stability. These are point mutations in the followingpositions: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195,M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396,R400, W439, R444, N445, K446, Q449, R458, N471 and N484, again asdefined via α-amylase AA560, i.e. also according to the numbering of theletter. Among these, the following variants are said to be particularlyadvantageous: Delta G184; Delta (R181-G182); Delta (D183-G184); R28N, K;S94K; R118K; N125A, R, K; N174D; R181Q, E, K; G186R; W189R, K; N195F;M202L, T; Y298H, F; N299A; K302R, S303Q, N306G, D, R, K; R310A, K, Q, E,H, D, N; N314D; R320K; H324K; E345R, D, K, N; Y396F; R400T, K; W439R;R444K; N445K, Q; K446N; Q449E; R458K; N471E and N484Q.

There is a very extensive prior art available on the improvement in thestability of α-amylases to very different influences by carrying outpoint mutations on the molecules in question. Said prior art will betreated here to an appropriate extent, taking into account only thedocuments directed to the enzymes and substantially dispensing withthose documents which describe again the use of the (previouslydescribed) enzymes in question in a large variety of industrial sectors:

The applications WO 91/00343 A2 and EP 409299 A2 describe variants of B.amyloliquefaciens α-amylase which have reduced stability andsubstitutions in, in some cases two or more of, positions 113, 114, 116,123, 163, 164, 166, 238, 316, 322, 345, 349, 356, 386, 394 and 398,including the particularly advantageous variant in which R123 has beenreplaced with other amino acids, in particular C.

The application WO 91/00353 A2 claims all α-amylase variants havingincreased thermostability, in which amino acid substitutions have beenperformed in positions 111, 133 and/or 149 (according to the numberingof B. licheniformis α-amylase); they include the specifically describedsubstitutions A111T, H133Y and T1491 which are said to be advantageous.

The application WO 94/02597 A1 claims all α-amylase variants in whichone or more methionine (M) residues have been replaced with, inprinciple, any other amino acid except C and M; among these, the newlyto be inserted amino acids L, T, A, G, S, I or D, particularly L, T, Aor G, are said to be advantageous; this is said to apply particularly tothe M residues in positions 197, 200 and 206 (according to the numberingof B. licheniformis α-amylase, i.e. termamyl), with the termamylvariants of A, G, T, V, Q, F, L, H, I, S, N, C and D in position 197being specifically described.

The application WO 94/18314 A1 claims α-amylase variants stabilized tooxidation which are obtained by deleting methionine (M), tryptophan (W),cysteine (C) or tyrosine (Y) residues of the precursor molecule orsubstituting other amino acids for said residues; among these, positionsM8, M15, M197, M256, M304, M366 and M438 (according to the numbering ofB. licheniformis α-amylase) are emphasized and the variants M197A, I, T,C, M15L, T, N, D, S, V, I and any variants of W138, including deletionthereof, are explicitly claimed. The substitutions of any possible aminoacids for M8A, M15 and M197, in particular M197L, M256A, M304L, M366 A,Y, M438A and W138F, Y, A, L, H and C, are specifically described.

The application WO 96/05295 A2 reveals corresponding variants, inparticular those containing the amino acid substitutions M15T, M197T,M15S, W138Y, for use in bleach-containing agents, partly in combinationwith one another.

U.S. Pat. No. 5,824,532 specifies the substitutions T, L, A, R, N, D, E,G, I, K, F, P, S, V, H, Q for M197 (according to the numbering of B.licheniformis α-amylase) or deletion thereof and also the substitutionsM15 L, T, N, D, S and V as those which can be used for stabilizingα-amylases to oxidation; in addition, a substitution of W138 is alsorecommended here.

U.S. Pat. No. 6,297,037 B1, of the same family, discloses among thesethe substitutions M197T, M15L, M15T, W138F and W138Y and combinationsthereof as particularly stable to oxidation, temperature and/or alkali.

The application WO 95/10603 A1 claims in principle any α-amylasevariants having improved washing or cleaning performance, on which adeletion, substitution or addition of a single amino acid has beenperformed, except M197A or M197T (according to the numbering of B.licheniformis α-amylase) as sole modification. Among these, the claimsemphasize variants in positions 1, 2, 3, 15, 17-35, 29-35, 51-58, 68,85, 88, 94-104, 121-136, 140, 142, 148, 152, 142-182, 172-178, 187, 209,217, 230, 233, 242, 246-251, 255, 260-269, 290-293, 314-320, 341, 360,369-383, 393, 398, 409, 416-421, 435, 450, 458-465. Of these, only thefollowing modifications are described experimentally, partially inmultiple combinations with one another: 1*, 2*, L3V, M15T, R23K, S29A,A30E, Y31H, A33S, E34D, H351, H68Q, S148N, M197I, N, S, T; A209V, L230I,V233A, R242P, E255P, T341P, S373P, Q374P, H450Y, E458D, P459T, V461K,N463G and E465D.

The application WO 95/35382 A2 claims any α-amylase variants having asimilarity of at least 70% identity to B. licheniformis α-amylase, inwhich variants amino acids in positions 104, 128, 187 and/or 188(according to the numbering of said α-amylase) have been replaced withany other amino acids. Among these variants, the substitutions N104D,V128E, S187D and N188D are particularly emphasized because they aredescribed in combination with one another and/or with othersubstitutions disclosed in the prior art as those which improve theenzymic properties regarding various aspects.

The application WO 96/23873 A1 describes variants of four differentα-amylases having properties, including also regarding stability, whichhave been altered compared with the starting enzyme. Among them thosevariants are claimed which have a reduced sensitivity to oxidation dueto an M, W, C or T having been replaced with a less oxidizable aminoacid. Specific mention is made here of the residues M9, M10, M105, M202,M208, M261, M309, M382, M430 and M440 (according to the numbering of B.licheniformis α-amylase), in particular in the substitution by L, andthe substitutions M202L, T, F, I, V.

The application WO 96/23874 A1 discloses numerous variants obtainable bypoint mutations. These include those having point mutations (in eachcase according to the numbering of B. licheniformis α-amylase) inpositions 236 (N236I, Y, F, L, V), 281 (H281F, I, L) and 273 (Y273F, W),which have relatively high activity at relatively high pH, and thosehaving point mutations in positions 61, 62, 67, 106, 145, 212, 151, 214,150, 143, 146, 241, 236, 7, 259, 284, 350, 343, 427 and 481 (L61W, V, F;Y62W; F67W; K106R, F, W; G145F, W; I212F, L, W, Y, R, K; S151 replacedwith any other amino acid, particularly F, W, I or L; R214W; Y150R, K;F143F; R146W; L241I, F, Y, W; I236L, F, W, Y; L7F, I, W; V259F, I, L;F284W; F350W; F343W; L427F, L, W and V481F, I, L, W), which haveincreased thermostability and/or elevated temperature optimum.

As a development of WO 96/23874 A1, the application WO 97/41213 A1claims any variants of α-amylases characterized by their similarity tothat of B. licheniformis and having the following mutations (in eachcase according to the numbering of B. licheniformis α-amylase): A181E,D, Q, N, V; I201 (replaced with a bulkier amino acid), including I201W,F, L; Y203Q; Q9K, L, E; F11R, K, E; E12Q; D100N, L; V101H, R, K, D, E,F; V102A, T; I103H, K; N104R, K, D; H105R, K, D, E, W, F; L196R, K, D,E, F, Y; I212R, K, D, E; L230H, K, I; A232G, H, F, S, V; V233D; K234L,E; I236R, K, N, H, D, E; L241R, K, D, E, F; A260S; W263H; Q264R, D, K,E; N265K, R, D; A269R, K, D, E; L270R, K, H, D, E; V283H, D; F284H;D285N, L; V286R, K, H, D, E; Y290R, E, K; V312R, K, D, E; F323H; D325N,N326K, H, D, L; H327Q, N, E, D, F; Q330L, E; G332D; Q333R, K, H, EL;S334A, V, T, L, I, D; L335G, A, S, T, N; E336R+R375E; T337D, K; T338D,E; T339D; Q360K, R, E; D365N; or G371D, R; having substitutions inpositions H68, H91, H247, R305, K306, H382, K389, H405, H406, H450 orR483; having the following mutations: H140Y; H142Y; H159Y; H140D+H142R;H140K+H142D; or H142Y+H156Y; having the deletion of three amino acids inthe subsequence from T369 to I377; having the substitution of thesubsequence from T369 to I377 by any of the following partial sequences:I-P-T-H-S-V; I-P-T-H-G-V; I-P-Q-Y-N-I; having substitutions in positionsR169 or R173, including R169I, L, F, T or R173I, L, F, T; having themutations H156D; I201F; I212F; A209L, T; or V2081; having substitutionsin positions N172, A181, N188, N190, H205, D207, A209, A210, E211, Q264or N265, including N172R, H, K; A181T; N188P; N190L, F; H205C; D207Y;A209L, T, V; A210S; E211Q; Q264A, E, L, K, S, T; N265A, S, T, Y; orQ264S+N265Y or H156Y+A181T+A209V; or in the form of further specialmultiple variants comprising at least five amino acid substitutions. Ineach case, some of these variants are said to have altered enzymicproperties, with inter alia also increased stability to deviating pH,elevated temperatures and oxidation being mentioned.

The application WO 98/26078 A1 describes stabilizing substitutions ordeletions in the B. licheniformis α-amylases, that is in positions A33,A52, S85, N96, H133, S148, A209, A269, A379 and A435 (according to thenumbering of B. licheniformis α-amylase), with the following variantsbeing specifically referred to: A33S, A52S, N96Q, H133Y, S148N, A209V,A269K, A379S and A435S. Among these, the substitutions A33S, A52S, N96Q,H133Y, S148N, A209V and A379S are described experimentally, in variouscombinations with further substitutions, namely M15T, D28N, N188S,A210S, T322A, G433D and I479T.

The European application EP 985731 A1 derived from the internationalapplication WO 98/44126 A1 describes the α-amylase from B. speciesKSM-AP 1378, which has been modified in position 202; particular mentionis made of the substitutions M202T, I, L, A, V, S, C. Accordingly, thesevariants have improved activity in the alkaline region and higherstability to oxidation. In contrast, the substitutions M9L, M105L (orI), M116D, M382L, M430L (or I) and M208Y are said to have had noincreased stability over the wild type molecule.

The application WO 99/02702 A1 claims α-amylase variants in which, forthe purpose of stabilization, an additional cystine bridge has beenintroduced by way of a point mutation; the amino acid substitutionsE119C/S130C and/or D124C/R127C (according to the numbering of B.licheniformis α-amylase) are said to be advantageous to this end, andthese modifications can apparently be combined with variations in otherpositions.

The application WO 99/09183 A1 describes and claims α-amylase variantsin which the amino acids A210, H405 and/or T412 (according to thenumbering of B. licheniformis α-amylase) have been replaced with otherones. Of these variants, the B. licheniformis α-amylase variantA210T/H405D/T412A is described in more detail.

The application WO 99/19467 A1 claims any α-amylase variants in which upto six of the following deletions or substitutions have been carried out(according to the numbering of α-amylase S707): (1.) R181*, G182*,T183*, G184*; (2.) N195, replaced with any other amino acid; (3.) V206,replaced with any other amino acid; (4.) E212, replaced with any otheramino acid; (5.) E216, replaced with any other amino acid and/or (6.)K269, replaced with any other amino acid; they are said to have alteredenzymic properties, including also improved stability. Variants whichare particularly stable to acid and calcium-independent and which aredistinguished experimentally, are N190F/Q264S of B. licheniformisα-amylase and I181*/G182*, I181*/G182*/N193F and I181*/G182*/N193F/E214Qof B. stearothermophilus α-amylase.

The application WO 99/23211 A1 claims any α-amylase variants in whichamino acids in positions 141, 142, 143, 144, 145, 146, 147, 148, 149,174, 181, 182, 183, 184, 185, 186, 189, 193, 195, 107, 108, 109, 166,167, 168, 169, 170, 171, 172, 173, 267, 268, 269, 270, 271, 272, 273,274, 275, 311, 346, 385, 456, 457, 458, 459, 460, 461, 462 and/or 463(according to the numbering of B. licheniformis α-amylase SP722) havebeen replaced with various other ones or have been deleted. Among these,the following point mutations are emphasized: K142R; S193P; N195F;K269R, Q; N270Y, R, D; K311R; E346Q, K385R; K458R; P459T; T461P; Q174*;R181Q, N, S; G182T, S, N; D183*; G184*; K185A, R, D, C, E, Q, G, H, I,L, M, N, F, P, S, T, W, Y, V; A186T, S, N, I, V, R; W189T, S, N, Q.These are said to have improved properties, inter alia with respect tostability to temperatures of between 10 and 60° C. and/or pH of between8 and 10.5.

The application WO 00/29560 A1 reveals α-amylase variants, in particularof hybrid amylases having point mutations in one or more positions onthe surface, whereby overall hydrophobicity is increased or the numberof side-chain methyl groups exposed to the medium are increased. This issaid to increase thermostability. The residues E376, S417, A420, S356and Y358 (according to the numbering of B. licheniformis α-amylase) arereferred to as preferred targets to this end, with the substitutionsE376K, S417T, A420Q, R; S356A and/or Y358F being preferred. They mayadditionally be combined with substitutions in positions K176, I201and/or H205, preferably K176R, I201F and/or H205N.

The application WO 01/34784 A1 describes variants of fungamyl(Aspergillus oryzae) α-amylase which per se has only lowthermostability, and claims any α-amylases which are at least 60%identical to Fungamyl and which according to this are understood asbeing a different group than the Termamyl-like α-amylases. Thesubstitutions are said to be in the regions 98-110, 150-160, 161-167,280-288, 448-455 and 468-475 (according to the numbering of A. oryzaeα-amylase); of these, only the substitution Q153S is experimentallydemonstrated to be stabilizing.

The application WO 02/31124 A2 specifies numerous point mutationsobtained by random mutagenesis of B. sp. KSM-K38 α-amylase. They aresaid to change the properties of the molecule, with the particularinfluences not being named in detail. The substitutions are as follows(according to the numbering of said amylase), it being possible for aplurality of substitutions also to occur together: G2P, A; M9I, L, F;H14Y; L15M, I, F, T; E16P; H26Y, Q, R, N; D27N, S, T; G48A, V, S, T;N49X; Q51X; A52X; D53E, Q, R; V54X; A58V, L, I, F; V73L, I, F; E84Q;G88X; D94X; N96Q; M103I, L, F; N104D; M/L107G, A, V, T, S, I, L, F;G108A; F111G, A, V, I, L, T; A114D, I, L, M, V, R; T125S; D128T, E;S130T, C; Y133F, H; W138F, Y; G140H, R, K, D, N; D142H, R, K, N; S144P;N148S; A149I; R156H, K, D, N; N161X; W165R; D166E; R168P; E171L, I, F;H173R, K, L; I173L; L174I, F; A178N, Q, R, K, H; N179G, A, T, S; T180N,Q, R, K, H; N181X; N183X; W184R, K; D187N, S, T; E188P, T, I, S; N190F;D194X; L197X; G198X; S199X; N200X; I201L, M, F, Y; D202X; F203L, I, F,M; S204X; H205X; E207Y, R; Q209V, L, I, F, M; E210X; E211Q; L212I, F;D214N, R, K, H; D221N; E222Q, T; D224N, Q; Y228F; L230I, F; I233A, V, L,F; K234N, Q; P237X; W239X; T241L, I, F, M; S242P, R; A252T; D253G, A, V,N; Q254K; D255N, Q, E, P; G260A; K264Q, S, T; D265N, Y; V267L, I, F, M;D275N, T; E276K; M277T, I, L, F; E280N, T, Q, S; M281H, I, L, F; V286X,preferably V286Y, L, I, F; Y290X; Y293H, F; S301G, A, D, K, E, R; R305A,K, Q, E, H, D, N; E314K, Q, R, S, T, H, N; A315K, R, S; I318L, M, F;T329S; E333Q; A340R, K, N, D, Q, E; D341P, T, S, Q, N; G356Q, E, S, T,A; S375P; A376S; K377L, I, F, M; M3801L, F; E383P, Q; L384I, F; D386N,Q, R, K, I, L; Q389K, R; Y399A, D, H; W403X; D404N; I405L, F; V406I, L,F, A, D; N427X; H441K, N, D, Q, E; R442Q; Q444E, K, R; A445V; Q448A;H453R, K, Q, N; A454S, T, P; G472R, N479Q, K, R; Q480K, R.Experimentally, however, only the following variants are disclosed:E84Q, N96D, A315S, A445V, G464N, N121D and N390H; further mention ismade of the possibility of performing the substitutions T125S, S144P,I173L, D210E, N393H, V408I, R442Q, N444H, Q448A and G464S. Specificeffects are not demonstrated.

All of these applications regarding point mutagenesis share the factthat the point of stability is also evaluated under the aspect of a goodperformance in the corresponding field of use. This is because thestability maintained during storage and usage of α-amylases, for examplewithin the context of detergent formulations, results in a highperformance or in a performance as constantly high as possible withcorresponding usage. They do not include any documents whichspecifically relate to the increase in stability during preparation,purification and formulation, i.e. processing of said α-amylases.

The purpose of increasing stability is also pursued by numerousapplications describing special enzyme stabilizers. These additionalingredients cause a protein and/or enzyme present in correspondingagents to be protected from damage such as, for example, inactivation,denaturation or decay, particularly during storage. Thus, reversibleprotease inhibitors form a group of stabilizers. Others, for examplepolyols, stabilize to physical influences such as freezing, for example.Other polymeric compounds such as acrylic polymers and/or polyamidesstabilize the enzyme preparation inter alia to pH fluctuations. Reducingagents and antioxidants increase stability of the enzymes to oxidativedecay.

Compounds of these kind are added to the enzymes both during applicationand in the course of their work-up, which is particularly important, ifa previously present stabilizer has been removed together with the othercontaminations in a component step of said workup, for example aprecipitation.

The prior art regarding the improvement in stability of α-amylases canbe summarized as follows: a multiplicity of α-amylase variants have beendeveloped via point mutagenesis, with the aim of these developmentshaving mainly been that of improving the performance of said α-amylasevariants. This category also includes those variants which have beenstabilized with regard to denaturing agents such as bleaches orsurfactants, since in these cases, the desired performance of the enzymeis optimized. In other cases, additional compounds which are overallreferred to as stabilizers are mainly used for increasing stability ormaintaining the physicochemical conformation.

A previously less regarded aspect in enzyme development is that ofstabilizing the molecules per se in such a way that they have increasedstability over the wild type molecule even during their workup. Anadditional advantageous effect thereof would be that this increasedstability should also benefit the intended later usage of the enzyme inquestion.

The necessity for this is particularly evident in the case ofα-amylases. At least some of these exhibit marked instability,especially during production and workup, which manifests itselfmacroscopically in a loss of activity. This applies in particular toprocess steps during which they are in aqueous solution, in particularat elevated temperature and at high (more alkaline) pH. As a result, theactivities in question are lost even during workup. The work-up processincludes all steps of industrial production, starting from isolating theenzyme in question, in particular the fermentation media common inbiotechnological production, via the following washing and separationsteps (for example by precipitation) and concentration up toformulation, for example granulation. However, the loss of activity mayalso occur during storage of α-amylase-containing agents or duringapplication, for example when used as active ingredient in washing orcleaning processes.

This problem can go as far as individual α-amylases, although they canbe produced and studied on a laboratory scale, refusing to be producedon an industrial scale with the aid of generally common methods. This isthen also referred to as said enzymes having low process stability orprocessivity, meaning a large variety of possible processings and uses.For example, Bacillus sp. A 7-7 (DSM 12368) α-amylase exhibits greaterinstability than the native B. licheniformis α-amylase. Approaches toeliminate said instability would only enable such enzymes in the firstplace to be accessible to production on an industrial scale and thus tothe large variety of fields of use in industrially relevant quantities.

SUMMARY OF THE INVENTION

It was therefore the object to stabilize α-amylases, in particular thatof Bacillus sp. A 7-7 (DSM 12368), per se in such a way that they have,compared to the starting molecule, increased stability to a loss ofactivity, in particular at elevated temperature and increased (morealkaline) pH.

In one partial aspect of said object, first a possible cause of thissusceptibility, which is based on the structure of the enzymes inquestion, had to be determined. Subsequently, said cause was to beanswered by suitable structural modifications.

Said structural modifications would firstly have an advantageous effecton the workup, i.e. industrial production, of said enzymes. Secondly,they should also be advantageous for the use of α-amylases, for examplein detergents and cleansers, because this should additionally beaccompanied by a constantly high activity during said use.

Particularly advantageous solutions to this object would therefore beconsidered to be those α-amylases which, besides said stability, exhibitfurther positive properties with regard to their intended use, inparticular with regard to their use in detergents and cleansers.

This applies especially to Bacillus sp. A 7-7 (DSM 12368) α-amylase.Preference is nonetheless given to those solutions which can betransferred to other α-amylases.

On the way to solve said object, the presence of particularlyhydrolysis-sensitive amino acid residues on the surface of α-amylases intheir native, correctly folded globular structure was contemplated as areason for the phenomenon of loss of activity in solution, also inaqueous solution and here in particular at alkaline pH, and of loss ofactivity at elevated temperature, which phenomenon is observed inparticular with α-amylases, and very particularly with certainα-amylases. Among said amino acid residues, the neutral, acid amidegroup-containing amino acid side chains, i.e. asparagine and glutamine(N and Q, respectively), rather than the ionizable, basic or acidicamino acid side chains (H, K, R or D and E), those containing a hydroxylgroup (S and T), the space-filling, cyclic or aromatic (H or F, Y, W),the sulfur-containing (C and M) or the aliphatic amino acid side chains(G, A, V, L and I), turned out to be the most effective starting points.The former appear to be hydrolyzed in particular at elevatedtemperatures and under alkaline conditions. Said hydrolysis of theseresidues then appears to destabilize the structure and thus thefunctionality of the entire enzyme.

A molecular-biological approach is pursued in order to solve the objectin question. The reason is that this influences the enzymic propertiesmediated by the mere amino acid sequence. In this connection, it wassurprisingly found that modifications, i.e. point mutations which areused to replace the amino acids N and Q present on the surface of theseα-amylases with other amino acids, stabilized α-amylases.

The amino acid residues present “on the surface of these molecules” canbe defined by the 3D structure of the globular enzyme protein, the“Conolly surface” or the “accessibility” value, i.e. solventaccessibility (see below).

Without wishing to be bound to this theory, it can be assumed that thesubstitution of one or more N or Q residues present on the surface ofα-amylases by other amino acid residues has less influence on themolecular structure than uncontrolled hydrolysis of the residue inquestion upon dissolving the enzyme in an aqueous, in particularalkaline, solvent and/or at elevated temperatures. As a result, from astatistical point of view, the majority of said enzymes is present intheir native conformation, whereby the preparation in question hassignificantly higher activity values, even at relatively long storageunder said conditions. This is beneficial both to isolation (for examplein a precipitation step) and storage (for example in solvents) and tothe usage.

This assumption is supported by the following comparison: B.licheniformis α-amylase has 18 N and 14 Q residues (i.e. 32 altogether)with an accessibility of at least 10% on its surface. B.amyloliquefaciens α-amylase has 22 N and 19 Q residues of this kind (41altogether), while Bacillus sp. A 7-7 (DSM 12368) α-amylase, asillustrated in Example 2 of the present application, has 41 N and 14 Qresidues of this kind (i.e. 55 in total). According to the teaching ofthe present application, there are corresponding possibilities ofimprovements here.

The object in question is consequently solved by α-amylase variantshaving at least one amino acid substitution over the starting molecule,whereby at least one asparagine (N) or glutamine (Q) residue on thesurface of said molecule has been replaced with A, C, F, G, H, I, K, L,M, P, R, S, T, V, W or Y.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Representation of the Conolly surface of Bacillus sp. A 7-7 (DSM12368) α-amylase; front view. The image was generated with the aid ofthe Swiss-Pdb viewer (Swiss Institute of Bioinfomatics;http://us.expasy.org/spdbv/; details: see text). Color coding: lightgray:asparagine residues; dark gray: glutamine residues.

FIG. 2: Representation of the Conolly surface of Bacillus sp. A 7-7 (DSM12368) α-amylase; rear view. Further information, see FIG. 1 and text.

FIG. 3: Alignment of Bacillus sp. A 7-7 (DSM 12368) α-amylase (SEQ IDNO. 2) with the most important other α-amylases of the prior art, ineach case across the region of the mature protein, i.e. positions 32 to516 according to SEQ ID NO. 2. Individual numbering can be carried outon the basis of the positional number indicated after the particularname of the first amino acid depicted in each case.

DETAILED DESCRIPTION

The amino acids in the present application are abbreviated using theinternationally common one- or three-letter code.

α-Amylases mean in accordance with the present application, asillustrated at the outset, the enzymes of class E.C. 3.2.1.1, whichhydrolyze internal α-1,4-glycosidic bonds of starch and starch-likepolymers with the formation of dextrins and β-1,6-branchedoligosaccharides. The invention can in principle be applied to any knownand still-to-be-found α-amylases, as long as they can be homologizedwith the α-amylase of Bacillus sp. A 7-7 (DSM 12368; SEQ ID NO. 2 of thepresent application). Such a homologization is depicted in FIG. 2 forthe industrially most important α-amylases and allows or facilitatesdetecting the amino acid positions according to the invention in theother amylases. The invention is preferably applied to those α-amylaseswhich are already employed successfully in industrial fields. Examplesof α-amylase established in the prior art are given in the introduction.

α-Amylase variants are those α-amylases which have been derived from aprecursor molecule by genetic modifications known per se. “Variant” isthe term at the protein level, which corresponds to “mutant” at thenucleic acid level. The precursor or starting molecules may be wild typeenzymes, i.e. those which are obtainable from natural sources. Thesehave been introduced by way of example at the outset. They may also beenzymes which are already variants per se, i.e. which have already beenmodified compared to the wild type molecules. They mean, for example,point mutants, those with changes in the amino acid sequence, overseveral positions or longer contiguous regions, or else hybrid moleculeswhich are composed of sections of various wild type α-amylases, whichcomplement each other. Wild type enzymes as well as mutants and hybridenzymes have been introduced by way of example at the outset. It ispossible, according to the invention, to further develop in principleany α-amylases. If the nucleic acids encoding them are known, this iscarried out by way of established methods of mutagenesis on the nucleicacids in question; if said nucleic acids are not known, it is possibleto derive, on the basis of the amino acid sequence, nucleic acidsequences coding therefor and to modify the latter accordingly.

Amino acid substitutions mean substitutions of one amino acid by anotheramino acid. According to the invention, such substitutions are indicatedwith reference to the positions in which the substitution takes place,where appropriate in combination with the amino acids in question in theinternationally customary one-letter code. “Substitution in position 83”means, for example, that a variant has a different amino acid in theposition which has position 83 in the sequence of a reference protein.Such substitutions are usually carried out at the DNA level by way ofmutations of individual base pairs (see above). “N83D” means, forexample, that the reference enzyme has the amino acid asparagine atposition 83, while the observed variant has the amino acid aspartic acidat the position homologizable therewith. “83D” means that any, i.e.usually a naturally predetermined, amino acid has been replaced withaspartic acid at a position corresponding to position 83; and “N83X”means that the amino acid asparagine in position 83 has been replacedwith principally any other amino acid.

In principle, the amino acid substitutions according to the inventionand specified by the present application are not limited to the factthat they are the only substitutions in which the variant in questiondiffers from the wild type molecule. It is known in the prior art thatthe advantageous properties of individual point mutations can complementeach other. An α-amylase optimized with respect to particular propertiessuch as, for example, calcium binding or stability to surfactants may bedeveloped according to the invention additionally by the substitutionspresented herein. Therefore, embodiments of the present inventioncomprise any variants which have, in addition to other substitutions,also the substitutions according to the invention, compared to the wildtype molecule.

A reference enzyme with respect to numbering of the positions, which maybe considered according to the invention, is Bacillus sp. A 7-7 (DSM12368) α-amylase whose nucleotide sequence is indicated in SEQ ID NO. 1,followed by the corresponding amino acid sequence in SEQ ID NO. 2. Asillustrated in Example 1 of the present application, this sequenceinformation has been corrected in comparison with the description in WO02/10356 A2 in two positions, and in this form corresponds, according tothe current knowledge, exactly to the sequence data obtainable from thedeposited strain DSM 12368 described in WO 02/10356.

Said correct amino acid sequence is also revealed in the in each casefirst line of the alignment depicted in FIG. 1, which has beenestablished only for the mature parts of the particular enzymes. This isjustified by the fact that in vivo only the mature portion (i.e. thatwith the positive numbering in SEQ ID NO. 2) is active as α-amylase. Forsome enzymes such as, for example, AA349 and AA560, the patentliterature (WO 00/60060 A2) indicates anyway merely the mature sequenceparts.

The surface of the enzyme comprises all those amino acids of thenatively folded enzyme which face the solvent. In Bacillus sp. A 7-7(DSM 12368) α-amylase, for example, these are the 407 amino acids listedin detail in Example 2.

The surface amino acids of other α-amylases may in principle be obtainedby using the alignment of FIG. 2. This is true only approximately,however. Highly conserved and structurally secured regions such asα-helices or β-sheets usually produce good agreement; in more flexibleregions, in particular loops, the comparison based on the alignment,i.e. the primary structure, is uncertain. In all cases (if known), thesecure X-ray structure, as it is actually deposited meanwhile for mostcommercially important α-amylases in generally accessible databases, isdecisive. Said databases are, for example, GenBank (National Center forBiotechnology Information NCBI, National Institutes of health, Bethesda,Md., USA) and Swiss-Prot (Geneva Bioinformatics (GeneBio) S. A., Geneva,Switzerland; http://www.genebio.com/sprot.html).

If the 3D (or tertiary) structure has not yet been determined for amolecule of interest, the former may be obtained via homology modeling.This method of predicting the structure of proteins whose crystalstructures have not yet been resolved assumes that proteins having asimilar primary structure also have similar secondary and tertiarystructures. The similarity of two protein sequences can be determinedvia a suitable algorithm, for example BLAST, FASTA or CLUSTAL. Suchalgorithms are likewise available via generally accessible proteindatabases; thus, for example, GenBank and Swiss-Prot have correspondinglinks. The RSCB protein database (accessible via Max-Delbrück-Zentrum inBerlin, Germany, via the Internet addresshttp://www.pdb.mdc-berlin.de/pdb; Aug. 24, 2004) enables the user tofind for a particular sequence crystal structures of related proteins byway of a FASTA search.

The possible calculations based thereupon, for example with the aid ofthe “Swiss-Pdb Viewer”, are described in the publication “SWISS-Modeland the Swiss-Pdb Viewer: An environment for comparative proteinmodeling” (1997) by N. Guex and M. C. Peitsch in Electrophoresis, volume18, pages 2714 to 2723. Said viewer is accessible free of charge via theInternet address http://us.expasy.org/spdbv/ (Aug. 24, 2004) of theorganization Swiss Institute of Bioinformatics (Central Administration,Bâtiment Ecole de Pharmacie- room 3041, Université de Lausanne, 1015Lausanne, Switzerland). The corresponding manual (SwissPDB ViewerManual) is available at the Internet addresshttp://us.expasy.org/spdbv/text/selmenu.htm (Aug. 24, 2004).Superimposition of said structures can establish a leader structure ontowhich the protein sequence of the α-amylase of interest is then modeled.The individual steps for this can be found in said user manual.

All of the depictions of the surface of an enzyme discussed herein, i.e.the special listing of the surface amino acids of Bacillus sp. A 7-7(DSM 12368) α-amylase (cf. FIG. 1) as well as the modeling approachesillustrated, are based on a calculation. This involves rolling by way ofcalculation a probe of 1.4 Å in size over the surface of said protein.All positions contacted thereby are included in the area of contact withsaid probe and thus in the surface; relatively narrow clefts which aretheoretically open toward the outside are not regarded here as part ofthe surface but are classed as belonging to the interior of themolecule. Using this approach produces the “Conolly surface” which wasfirst described by M. L. Connolly in the article “Solvent-AccessibleSurfaces of Proteins and Nucleic Acids” (1983) in Science, volume 221,709-713. This acknowledged method is familiar to the skilled worker andis used according to the invention for defining the surface ofα-amylases.

In this way, the mentioned 407 surface amino acids were produced forBacillus sp. A 7-7 (DSM 12368) α-amylase, which are listed in detail inExample 2.

They include, as is furthermore depicted in Example 2, for Bacillus sp.A 7-7 (DSM 12368) α-amylase the following 44 asparagine residues as asubset of the previously determined 407 surface amino acid residues:

N6 (12), N19 (28), N22 (28), N25 (16), N33 (28), N54 (35), N70 (38), N83(44), N95 (19), N106 (1), N123 (19), N126 (18), N128 (31), N150 (39),N154 (41), N175 (20), N195 (24), N197 (3), N215 (27), N219 (32), N226(40), N255 (42), N260 (49), N270 (38), N277 (24), N283 (40), N285 (27),N296 (15), N299 (23), N306 (51), N314 (35), N331 (6), N402 (16), N409(11), N418 (25), N423 (40), N437 (37), N445 (41), N457 (29), N465 (22),N471 (20), N475 (25), N484 (12), N485 (38);

and the following 17 glutamine residues:

Q11 (4), Q53 (12), Q71 (5), Q84 (24), Q86 (18), Q98 (29), Q129 (31),Q169 (26), Q172 (53), Q174 (44), Q311 (35), Q319 (38), Q335 (3), Q361(36), Q394 (20), Q401 (14), Q449 (31).

The illustrations of FIGS. 1 and 2 depict these N and Q residues forBacillus sp. A 7-7 (DSM 12368) α-amylase. As can be seen, they aredistributed in an irregular arrangement over the entire molecularsurface.

Said residues may be replaced according to the invention in principlewith any other amino acid residues, unless they are N, Q, D or E. It isobvious that mutual conversion of N to Q or vice versa is not sensible.The exclusion of D and E is based on the fact that these free acidgroups which correspond chemically to the acid amide residues N and Qare likewise susceptible to hydrolysis. It is therefore recommendedaccording to the invention to substitute the remaining natural aminoacids, i.e. A, C, F, G, H, I, K, L, M, P, R, S, T, V, W or Y.

For information purposes as to which substitution can be chosen in eachcase, reference may be made here to the alignment of FIG. 3, withoutwishing to restrict hereby the substitutions referred to as preferredhereinbelow. N or Q residues are also present in many of said positionsdetermined on the basis of Bacillus sp. A 7-7 (DSM 12368) α-amylase inother α-amylases. Biochemically mostly useful alternatives thereof arerevealed by the amino acid sequences of the other α-amylases which donot have N or Q here. Thus, conspicuously, there is no methionineanywhere in a position homologous to a surface asparagine or glutamine.Very rarely, a C, and only slightly more frequently long-chain aliphaticamino acids, can be found there. In order to decide which amino acidscan advantageously replace an N or Q, it is advisable to use saidalignment for information, according to which usually a short-chainand/or polar residue should be preferred over said rare residues.

The success of this amino acid substitution according to the inventioncan be checked by way of solvent stability of the α-amylase variants inquestion, as illustrated in Example 4. Said variants have compared tothe particular starting enzymes, i.e. the wild type enzymes or thosewhich expressly do not have the substitutions according to theinvention, significantly better activities after a certain incubationtime in an appropriate medium. The application WO 02/10356 A2 describesa suitable activity assay. Said comparison is particularly meaningful ifcomparatively unfavorable basic conditions prevail during incubation,for example a pH higher than the pH optimal for stability or atemperature above the temperature optimum of the non-inventive variant.The reason is, as illustrated, that these are the situations to whichthe molecule in question should be particularly stabilized.Statistically, increased activities can be attributed here to a largernumber of α-amylase molecules which have survived said unfavorableconditions unharmed.

In a preferred embodiment, the α-amylase variants of the invention arethose wherein N has been replaced with A, G, K, R, S or T, preferablywith S or T, and Q has been replaced with A, G, I, K, R, S or T,preferably with S or T.

Of the 16 remaining amino acids, the space-filling aliphatic,sulfur-containing, aromatic or cyclic amino acids are preferably notused for substitution because they could cause undesired hydrophobiceffects or would introduce additional chemically active groups.Preferably P is also not used because it is usually associated withconsiderable structural modifications. Thus, for N, preferably the aminoacids containing small aliphatic groups, i.e. the neutral amino acids Aand G, the usually charged, basic amino acids K and R, and the polarresidues of S and T, remain in order to implement the present invention.The same applies to Q, with the substitution with I having likewiseproved to be advantageous.

In the individual case, it is recommended to select in each case themost suitable of said possibilities. In most cases, it has provedadvantageous to introduce either of the two amino acids S and T in placeof N or Q. Without wishing to be bound to this theory, it may besuspected that this is due to the chemical properties of said residues,which do not deviate very much from one another. S and T are like N andQ neutral side chains with medium space requirement; the latter islikely to be important for correct folding of the enzyme. Moreover, bothside chains are polar, with the values for S and T, at +5.1 and +4.9,respectively, being lower than, but still in the same order of magnitudeas, those for N and Q, at +9.7 and +9.4, respectively. This isespecially important against the background that these are surface aminoacids which do not protrude into the hydrophobic, apolar molecularinterior, but are in contact with the mainly hydrophilic, polarenvironment.

In a preferred embodiment, α-amylase variants of the invention are thosein which said amino acid residue has an accessibility of at least 10%,preferably at least 20%, particularly preferably at least 30%, prior toamino acid substitution, wherein said accessibility of the amino acidresidue in question is calculated on a scale from 0% (not accessible tothe solvent) to 100% (present in a hypothetical pentapeptide, GGXGG).This includes accordingly all intermediate integers and fractions;increasingly preferred embodiments are characterized by increasingaccessibility values, as can be found in the above information.

Accessibility of an amino acid residue means according to the invention,how well said residue is accessible to the surrounding solvent (usuallywater), with the molecule being in its natural conformation.Determination of this physicochemical property is based on a scale from0 to 100%, with a value of 0% accessibility meaning that the residue inquestion is not accessible to the solvent, and 100% being theaccessibility possible in a hypothetical pentapeptide GGXGG.

According to the invention, this molecular property is reflected in thatan exposed amino acid residue which stands out from the surface contactsother molecules more readily than one which is less accessible to thesolvent. Such residues are therefore preferred targets for hydrolysis,for example by nucleophiles such as hydroxyl ions. Consequently, saidresidues are also preferred targets for protecting the molecule via thepresent invention.

Using the abovementioned SwissPDB Viewer enables these values also to becalculated for each surface amino acid of a globular protein.Accessibility of the amino acids is calculated there via the menu item“Select-->Accessible aa”, followed by entering the accessibility inpercent. Subsequently, the corresponding amino acids are selected andcan be displayed by pressing the “Return” key. In addition, it ispossible here to automate said calculation via a self-build program, andthis is particularly recommended, if more amino acids are to be includedin said calculation.

As illustrated in Example 2, the following 41 N and 14 Q residues weredetermined for Bacillus sp. A 7-7 (DSM 12368) α-amylase, whoseaccessibility determinable in this way is at least 10%, and is indicatedin % in brackets following the positions listed:

N6 (12), N19 (28), N22 (28), N25 (16), N33 (28), N54 (35), N70 (38), N83(44), N95 (19), N123 (19), N126 (18), N128 (31), N150 (39), N154 (41),N175 (20), N195 (24), N215 (27), N219 (32), N226 (40), N255 (42), N260(49), N270 (38), N277 (24), N283 (40), N285 (27), N296 (15), N299 (23),N306 (51), N314 (35), N402 (16), N409 (11), N418 (25), N423 (40), N437(37), N445 (41), N457 (29), N465 (22), N471 (20), N475 (25), N484 (12),N485 (38);

and, respectively:

Q53 (12), Q84 (24), Q86 (18), Q98 (29), Q129 (31), Q169 (26), Q172 (53),Q174 (44), Q311 (35), Q319 (38), Q361 (36), Q394 (20), Q401 (14), Q449(31).

Of these, the following 32 and 11 have thus an accessibility of at least20%:

N19 (28), N22 (28), N33 (28), N54 (35), N70 (38), N83 (44), N128 (31),N150 (39), N154 (41), N175 (20), N195 (24), N215 (27), N219 (32), N226(40), N255 (42), N260 (49), N270 (38), N277 (24), N283 (40), N285 (27),N299 (23), N306 (51), N314 (35), N418 (25), N423 (40), N437 (37), N445(41), N457 (29), N465 (22), N471 (20), N475 (25), N485 (38);

and, respectively:

Q84 (24), Q98 (29), Q129 (31), Q169 (26), Q172 (53), Q174 (44), Q311(35), Q319 (38), Q361 (36), Q394 (20), Q449 (31).

Of these, the following 18 and 7 thus have an accessibility of at least30%:

N54 (35), N70 (38), N83 (44), N128 (31), N150 (39), N154 (41), N219(32), N226 (40), N255 (42), N260 (49), N270 (38), N283 (40), N306 (51),N314 (35), N423 (40), N437 (37), N445 (41), N485 (38);

and, respectively:

Q129 (31), Q172 (53), Q174 (44), Q311 (35), Q319 (38), Q361 (36), Q449(31).

This grouping is used merely for illustration. According to theinvention, the residues in question are increasingly preferred withincreasing accessibility because they are increasingly exposed to thesolvent and can therefore be hydrolyzed all the more readily.

An application to other α-amylases is possible by way of approximationvia an alignment such as in FIG. 3. However, as illustrated previously,the actual three dimensional structure is decisive, on the basis ofwhich the appropriate calculations of actual charge distribution andsolvent accessibility can be carried out, as demonstrated for Bacillussp. A 7-7 (DSM 12368) α-amylase in the example.

In a further preferred embodiment, α-amylase variants of the inventionare those α-amylase variants, wherein two or more of said amino acidsubstitutions have been carried out, preferably from 2 to 10,particularly preferably from 3 to 8, very particularly preferably from 4to 6.

According to the invention, each individual substitution according tothe invention is assumed to cause on its own an advantageous effect. Inprinciple, however, an additivity of these advantageous effects of allamino acid substitutions according to the invention cannot be assumed.Thus, in the individual case, it is recommended to carry out onemutation according to the invention after the other and to check whetherthis has in each case achieved an improvement in stability. Since ineach case a certain improvement (albeit not necessarily an additive oreven synergistic one) can be expected at least in principle, preferablymore than one substitution according to the invention is carried out.

Due to the nature of the enzymes, this procedure also has an individualupper limit which must be determined experimentally, where appropriate.The possible upper limit for this comprises in principle nearly all Nand Q residues located on the surface of the molecule. The preferredupper limit of ten substitutions, set at this point, is firstly stillachievable technically. Secondly, each mutation including thoseaccording to the invention, is accompanied by changes in the molecularproperties. An example of this are the polarity values indicated abovefor the particularly preferred substitutions with S or T. It can beassumed that, for the whole molecule, a too drastic decrease in allpolarities is in turn related to an increase in instability. This toojustifies the stipulation of determining the particular optimumexperimentally.

At least for Bacillus sp. A 7-7 (DSM12368) α-amylase, the limitsindicated have proved to be advantageous in several respects, withregard to both efficacy and achievability. A similar result can beexpected for the α-amylases homologizable therewith, for example thoseof FIG. 3 and in particular those which have likewise N or Q in thehomologous positions.

In a further preferred embodiment, α-amylase variants of the inventionare those α-amylase variants, wherein the starting molecule is any ofthe following α-amylases: α-amylase from Bacillus sp. A 7-7 (DSM 12368),α-amylase from Bacillus sp. #707, α-amylase from Bacillus sp.KSM-AP1378, α-amylase from Bacillus KSM-K38, α-amylase from B.amyloliquefaciens, α-amylase from B. licheniformis, α-amylase fromBacillus sp. MK716, α-amylase from Bacillus sp. TS-23, α-amylase from B.stearothermophilus, α-amylase from B. agaradherens, a cyclodextringlucanotransferase (CGTase) from B. agaradherens, in particular from B.agaradherens (DSM 9948), or a hybrid amylase therefrom and/or anα-amylase derived therefrom by mutagenesis of single or multiple aminoacids.

As illustrated at the outset, numerous α-amylases are established in theprior art for the use for various industrial purposes. These include,for example, fungal and bacterial enzymes. The present invention may inprinciple be applied to all of these α-amylases.

Those α-amylases of Gram-positive bacteria, in particular of the genusBacillus, which are adapted to an alkaline environment, are particularlycommon for industrial purposes. This is because they have favorableproperties for these fields of use from the outset. Accordingly, thepresent invention is directed in particular to the α-amylases which canbe obtained naturally from said species. These include in particular thefollowing:

Bacillus sp. A 7-7 (DSM 12368) α-amylase, disclosed in WO 02/10356 A2and the present application;

Bacillus sp. #707 α-amylase, disclosed in the publication “Nucleotidesequence of the maltohexaose-producing amylase gene from an alkalophilicBacillus sp. #707 and structural similarity to liquefying typeα-amylases” (1988) by Tsukamoto et al., Biochem. Biophys. Res. Comm.,volume 151(1), pages 25-31;

Bacillus sp. KSM-AP1378 α-amylase whose amino acid sequence (togetherwith point mutants) has been disclosed in EP 985731 A1;

Bacillus KSM-K38 α-amylase, disclosed in EP 1022334 A2;

B. amyloliquefaciens α-amylase (commercial product BAN® from Novozymes);

B. licheniformis α-amylase (commercial product Termamyl® fromNovozymes),

Bacillus sp. MK716 α-amylase whose sequence has been deposited undernumber AAB18785 in the database of the National Center for BiotechnologyInformation, National Library of Medicine, Building 38A, Bethesda, Md.20894, USA (GenBank; accessible at http:www.ncbi.nlm.nih.gov);

Bacillus sp. TS-23 α-amylase, disclosed in the publication: “Productionand property of raw-starch-degrading Amylase from the thermophilic andalkaliphilic Bacillus sp. TS-23” (1998) by Lin et al. in Biotechnol.Appl. Biochem., volume 28(1), pages 61 to 68; and

B. stearothermophilus α-amylase (commercial product Novamyl® fromNovozymes).

The amino acid sequences comprising the in each case mature regions ofthese enzymes are compiled in FIG. 2.

The present invention further relates to B. agaradherens α-amylase andtwo B. agaradherens cyclodextrin glucanotransferases (CGTases), whichare described in two international applications: WO 02/06508 A2 and WO02/44350 A2, the latter being formed by the deposited microorganism B.agaradherens (DSM 9948) described in the application in question andbeing preferred. The part of the CGTase molecule, which can behomologized with the previously described α-amylases, is in each casemodified according to the invention.

The starting enzyme to be introduced to the invention may also be ahybrid amylase of the α-amylases just mentioned. These are revealed, asalready mentioned at the outset, for example by the applications WO96/23784 A1 (hybrids of the α-amylases of Bacillus licheniformis, B.amyloloiquefaciens and B. stearothermophilus) and WO 03/014358 A2(special hybrid amylases of Bacillus licheniformis and B.amyloliquefaciens). The latter will be discussed in more detailhereinbelow.

In addition, the prior art describes numerous α-amylase variants derivedby mutagenesis of single or multiple amino acids of said α-amylases. Thepresent invention may likewise be applied to all of these, and therespective effects can be assumed in principle to be additive. Thus itshould be possible, by carrying out a mutation of the invention, toimprove an α-amylase variant which is particularly powerful in a specialfield of application, owing to a particular amino acid substitution,also in its tendency to aggregate, in addition to the former aspect ofits performance. These are thus preferably amino acid variations forwhich a corresponding advantage has been described previously.

In this connection, it does not matter in principle, in which order thesubstitutions in question have been carried out, i.e. whether acorresponding point mutant is further developed according to theinvention or initially a variant of the invention is generated, forexample, from a wild type molecule and is then further developedaccording to other teachings which can be found in the prior art. It isalso possible to carry out a plurality of substitutions, for examplethose according to the invention and others together, in a singlemutagenesis mix simultaneously. This situation is given, for example, ifan α-amylase is further developed using randomly acting mutagenesisprocesses, for example by means of mutagenizing agents or shuffling.This applies to any type of modification of the enzymes in question, inparticular to point mutations which in principle act independently ofone another.

Particularly powerful α-amylases obtainable by point mutations arerevealed, for example, by the application WO 00/60060 A2 whichdescribes, on the basis of the AA560 amylase (the same numbering as themature protein in SEQ ID NO. 2), numerous mutations in positions 181,182, 183, 184, 195, 206, 212, 216, and 269. This application can beconsidered a development of WO 96/23873 A1 which had previouslydescribed the possibility of point mutagenesis in positions 180, 181,182, 183, 184 and 185 in order to improve performance. The applicationWO 00/60059 A2 specifies further improvements of enzymes referred totherein as Termamyl-like amylases; according to this, positions 13, 48,49, 50, 51, 52, 53, 54, 57, 107, 108, 111, 168 and 197 (according to thenumbering of B. licheniformis α-amylase) are said to be suitablestarting points for this. All of these α-amylases which are improvedwith respect to their activity and/or performance via these positionsand in particular according to said applications, are, for the purposesof the present application, starting molecules particularly preferredaccording to the invention.

In particularly preferred embodiments, the starting molecule is any ofthe following α-amylases: α-amylase from Bacillus sp. A 7-7 (DSM 12368),cyclodextrin glucanotransferase from B. agaradherens (DSM 9948) or ahybrid amylase of the α-amylases from B. amyloliquefaciens and from B.licheniformis, preferably a hybrid amylase AL34, AL76, AL112, AL256,ALA34-84, LAL19-153 or LAL19-433.

Thus the invention described herein is illustrated in the examples ofthe present application in particular on the basis of Bacillus sp. A 7-7(DSM 12368) α-amylase whose most likely correct DNA and amino acidsequences are indicated in the sequence listing of the presentapplication. This wild type enzyme has the advantages illustrated in theapplication WO 02/10356 A2 with regard to a use in detergents andcleansers over other established α-amylases. Another particularlypreferred starting molecule, namely B. agaradherens (DSM 9948)cyclodextrin glucanotransferase, has already been mentioned above andlikewise makes particular contributions to the overall performance ofdetergents and cleansers over other enzymes, as described in theapplication WO 02/44350 A2.

The application WO 03/014358 describes hybrid amylases of the α-amylasesB. amyloliquefaciens and B. licheniformis. Of these, the moleculesreferred to by the acronyms AL34, AL76, AL112, AL256, ALA34-84,LAL19-153 or LAL19-433 have exhibited particularly advantageousperformances when used in detergents and cleansers and are thereforeparticularly preferably also introduced to the present invention, i.e.are mutated according to the invention. The particular names refer tothe elements of which they are composed in each case, as seen from the Nterminus, wherein A is the B. amyloliquefaciens α-amylase and L is theB. licheniformis α-amylase and the subsequent number indicates thejunction between the first and second amino acid sequences. Furtherdetails on this can be found in said application.

In a further preferred embodiment, α-amylase variants of the inventionare those α-amylase variants, wherein the starting molecule is anα-amylase whose amino acid sequence is at least 96% identical,preferably 98%, particularly preferably 100%, identical to the aminoacid sequence indicated in SEQ ID NO. 2 in positions +1 to 484.

This Bacillus sp. A 7-7 (DSM 12368) α-amylase is described in detail inthe international application WO 02/10356 A2 which has already beenmentioned several times. It should be noted in this context that thenucleotide sequence and the amino acid sequence derived therefrom of theα-amylase obtainable from said strain have most likely the sequencesindicated in SEQ ID NO. 1 and 2, respectively, of the presentapplication. This represents a correaction of the sequence indicated inthe application WO 02/10356 A2. The experiments described in thatapplication have also been carried out using the native α-amylaseobtainable from said strain.

The nucleotide sequence according to SEQ ID NO. 1 enables the skilledworker immediately to carry out mutations according to the invention. Anα-amylase-encoding DNA which has been prepared according to the sequencelisting of WO 02/10356 A2 rather than by using the depositedmicroorganism, may be converted with the aid of simple point mutagenesismethods, as they are also mentioned in the examples of the presentapplication, to a nucleotide sequence according to SEQ ID NO. 1 of thepresent application.

The possibilities of culturing the corresponding microorganism,purification and enzymic characterization of the wild type α-amylasehave likewise been disclosed in that application and are summarizedagain in Example 1 of the present application. Variants according to theinvention of this enzyme can in principle be produced and purified inthe same way, with the differences introduced according to theinvention, for example in view of the isoelectric point (IEP), having tobe taken into account.

Further preference is given to those α-amylase variants according to theinvention, wherein the starting molecule is an α-amylase variantcontaining, with increasing preference, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or11 additional point mutations.

The sequences indicated in SEQ ID NO. 1 and 2 differ in each case in twopositions from the information in WO 02/10356 A2, as illustrated inExample 1. According to current knowledge, they represent the bestdescriptions of Bacillus sp. A 7-7 (DSM 12368) α-amylase and aretherefore particularly preferred starting points for introducing aminoacid substitutions of the invention.

According to the above, it may further be assumed that in principle anyα-amylase can be improved by individual point mutations, i.e. deletions,insertions or substitutions of individual amino acids, with respect toother aspects. Thus, as already illustrated at the outset, theapplications WO 00/22103 A1, WO 96/23873 A1, WO 00/60060 A2 and WO01/66712 A2 reveal α-amylase variants which have been mutagenized inindividual positions and which have been improved in each case withrespect to special aspects. Since some of said variants have highhomology to said amylase depicted in SEQ ID NO. 2, i.e. they can bereferred to as highly related, the substitutions specified therein canbe expected to be applicable also to these molecules and, analogously,also to other α-amylases, with the same advantageous effects. Furtherpossible modifications can be found in the prior art as summarized, forexample, at the outset of the present application.

Furthermore preferred embodiments of the present invention are α-amylasevariants according to the invention, having the additional pointmutations in one or more of the following positions: 5, 167, 170, 177,202, 204, 271, 330, 377, 385 and 445, according to the numbering of themature protein according to SEQ ID NO. 2.

Thus, the German application DE 10309803.8 which has not beenprepublished reveals that point mutations may be carried out inpositions −19, 5, 167, 170, 177, 204, 271, 330, 377, 385 and/or 445 (or13, 32, 194, 197, 203, 230, 297, 356, 406, 414 and 474 according to thenumbering of the unprocessed B. amyloliquefaciens α-amylase), in orderto improve the alkali activity of α-amylases. Preference is thereforegiven to applying the present invention to these already improvedmolecules.

As already illustrated at the outset, the application WO 94/18314 A1reveals α-amylase variants stabilized to oxidation, including especiallyin position 197 (according to the numbering of B. licheniformisα-amylase) corresponding to position 202 of the α-amylase according toSEQ ID NO. 2. Variants according to the invention may be stabilizedadditionally to oxidation by the substitutions mentioned therein,especially in position 202. Said substitution is of particular interestalso because M202 in Bacillus sp. A 7-7 (DSM 12368) α-amylase is theonly methionine residue which is more than 10% accessible to thesolvent, having an accessibility value of 30% (cf. Example 2). Thisexplains its particular sensitivity to oxidation and the connection withthe present invention.

Preferred embodiments thereof are those α-amylase variants, wherein thepoint mutations in the starting enzymes are as follows: 5A, 167R, 170P,177L, 202L, 204V, 271D, 330D, 377R, 385S and/or 445Q.

Thus, the mentioned application DE 10309803.8 describes the sequencevariations −19P, 5A, 167R, 170P, 177L, 204V, 271D, 330D, 377R, 385Sand/or 445Q (or, according to the numbering of the unprocessed B.licheniformis α-amylase, the substitutions to give 13P, 32A, 194R, 197P,203L, 230V, 297D, 356D, 406R, 414S and 474Q) as preferred substitutions.

WO 94/18314 A1 illustrates in particular the oxidation-stabilizingaction of the substitution M197L corresponding to the amino acidsubstitution M202L according to SEQ ID NO. 2. Variants according to theinvention may therefore be stabilized additionally to oxidationparticularly by this point mutation.

Another solution to the object in question and thus another subjectmatter of the present invention are methods of increasing the stabilityof an α-amylase to solvent-exerted hydrolysis, whereby at least oneasparagine (N) or glutamine (Q) residue on the surface of said moleculehas been replaced with A, C, F, G, H, I, K, L, M, P, R, S, T, V, W or Y.

As discussed, this is because according to the invention a considerableportion of the loss of activity of α-amylase-containing solutions can beattributed to the hydrolysis of the sensitive surface amino acidsasparagine (N) and glutamine (Q). This is understood as being astability aspect. Any method that counteracts the hydrolysis of saidamino acid residues is therefore one that increases the stability of theenzyme preparation in question with respect to its overall activity.

The approach according to the invention in order to solve said problemconsists of, as likewise illustrated above, introducing another aminoacid at their site, for which in principle any other amino acid otherthan Q and N, D or E is suitable.

Methods of preparing enzyme variants are known per se to abiotechnologist. He will use in particular the nucleic acids coding forthe starting enzymes in question, which is mutated in the correspondingcodon according to the amino acid to be introduced.

The principle of this is also illustrated in Example 3: primers whichcover the region to be mutated and contain the mutation to be introduced(mismatch primers) are synthesized, for example, with the aid of theQuikChange kit (Stratagene, cat. No. 200518) according to thecorresponding protocol. The primer sequences are designed on the basisof the corresponding nucleotide sequence, taking into account theuniversal genetic code. According to this principle, an expressionvector containing the α-amylase sequence is suitably mutagenizedaccordingly and transformed into an expression strain suitable forexpressing said amylase, using generally known methods. Since thevariant usually has only a few substitutions compared to the startingmolecule, the systems established for said starting molecule can be usedfor information when choosing the expression system, culturing andwork-up methods. The fact that the biophysical properties of α-amylasesare altered according to the invention must be taken into account here.A corresponding change in the IEP may be checked, for example, with theaid of isoelectric focusing.

Preference is given to methods of the invention for increasing thestability of an α-amylase to solvent-exerted hydrolysis at elevatedtemperatures or high pH.

This is, as discussed, because the described instability can be observedin particular under these conditions. This may be explained by the factthat chemical reactions are generally faster at higher temperatures andbiopolymers such as proteins are more readily denatured at highertemperatures, but this thesis should not be regarded as binding. On theother hand, the hydroxyl ion which is increasingly abundant at alkalinepH is a very reactive nucleophile, in particular toward the carboxylgroup of these same acid amides.

Other than that, the comments made above regarding the particularenzymes apply accordingly. This also applies accordingly to thepreferred embodiments, i.e. to those methods of the invention which areused to obtain the above-described variants according to the invention.

According to the above, preferred methods of the invention are those,wherein N has been replaced with A, G, K, R, S or T, preferably S or T,or Q has been replaced with A, G, I, K, R, S or T, preferably S or T.

According to the above, further preferred methods of the invention arethose, wherein said amino acid residue has an accessibility of at least10%, preferably at least 20%, particularly preferably at least 30%,prior to amino acid substitution, wherein said accessibility of theamino acid residue in question is calculated on a scale from 0% (notaccessible to the solvent) to 100% (present in a hypotheticalpentapeptide, GGXGG).

According to the above, further preferred methods of the invention arethose, wherein a plurality of said amino acid substitutions have beencarried out, preferably from 2 to 10, particularly preferably from 3 to8, very particularly preferably from 4 to 6.

The order of the individual substitutions is in principle unimportanthere because the properties of the finally obtained molecule aredecisive.

According to the above, further preferred methods of the invention arethose, wherein the starting molecule is any of the following α-amylases:α-amylase from Bacillus sp. A 7-7 (DSM 12368), α-amylase from Bacillussp. #707, α-amylase from Bacillus sp. KSM-AP1378, α-amylase fromBacillus KSM-K38, α-amylase from B. amyloliquefaciens, α-amylase from B.licheniformis, α-amylase from Bacillus sp. MK716, α-amylase fromBacillus sp. TS-23, α-amylase from B. stearothermophilus, α-amylase fromB. agaradherens, a cyclodextrin glucanotransferase (CGTase) from B.agaradherens, in particular from B. agaradherens (DSM 9948), or a hybridamylase therefrom and/or an α-amylase derived therefrom by mutagenesisof single or multiple amino acids.

Among these, preference is given according to the above two methods,wherein the starting molecule is any of the following α-amylases:α-amylase from Bacillus sp. A 7-7 (DSM 12368), cyclodextringlucanotransferase from B. agaradherens (DSM 9948) or a hybrid amylaseof the α-amylases from B. amyloliquefaciens and from B. licheniformis,preferably a hybrid amylase AL34, AL76, AL112, AL256, ALA34-84,LAL19-153 or LAL19-433.

According to the above, further preferred methods of the invention arethose, wherein the starting molecule is an α-amylase whose amino acidsequence is at least 96% identical, preferably 98%, particularlypreferably 100%, identical to the amino acid sequence indicated in SEQID NO. 2 in positions +1 to 485.

Among these, preference is given according to the above to methods,wherein the starting molecule is an α-amylase variant containing, withincreasing preference, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 additionalpoint mutations.

According to the above, further preferred methods of the invention arethose, wherein the introduced α-amylase is an α-amylase variant havingthe additional point mutations in one or more of the followingpositions: 5, 167, 170, 177, 202, 204, 271, 330, 377, 385 and 445,according to the numbering of the mature protein according to SEQ ID NO.2.

At this point, it should be noted once again that it does not matter inprinciple for mutants which can be used as starting molecules accordingto the invention, as to whether the amino acid substitution according tothe invention takes place after or before introducing the othersubstitutions disclosed in the prior art.

Among said methods, preference is given according to the above tomethods, wherein the point mutations are as follows: 5A, 167R, 170P,177L, 202L, 204V, 271D, 330D, 377R, 385S and/or 445Q.

The present invention further relates to nucleic acids coding for any ofthe above-described α-amylase variants according to the invention.

Said nucleic acids are understood as meaning both RNA and DNA moleculesand also DNA analogs, both the coding and the codogenic strand, that isin every reading frame, because it is possible, for example, to utilizethe teaching associated with the invention, in order to regulatecorresponding α-amylases via an interfering corresponding RNA or toincrease the lifetime of said genetic information by converting it to aDNA analog which is more slowly degradable in vivo. To a certain extent,said nucleic acids present the molecular-biological dimension to thepresent invention, as is discernible from the subject matters of theinvention set forth hereinbelow. According to the above, they arepreferred accordingly.

Said nucleic acids are understood as meaning preferably DNA moleculesbecause they can be used for preparing and/or, where appropriate,further modifying the α-amylase variants of the invention bymolecular-biological methods known per se. The nucleotide sequences ofthe abovementioned α-amylases established in the prior art are depositedin well-known databases (for example Genbank or Swissport; see above) orare revealed in said publications. These sequences are accordinglypreferred starting points for introducing point mutations according tothe invention.

Starting points for said nucleic acid may also be the sequenceinformation indicated in the sequences, in particular for derivatives ofBacillus sp. A 7-7 (DSM 12368) α-amylase. Another alternative consistsof preparing total DNA of particular microorganisms considered for thisand isolating the endogenous α-amylase genes by PCR. Primers which maybe employed in principle are the 5′ and 3′ sequence sections which canlikewise be read from SEQ ID NO. 1.

The pure protein-encoding sections may, for example, also be mutagenizedby a PCR, i.e. their sequence may be modified according to theinvention. To this end, a PCR with a corresponding statistical errorrate is carried out, the PCR product is cloned and the introducedmutations are verified by subsequent sequencing.

Preference is given here to a nucleic acid which derives from a nucleicacid according to SEQ ID NO. 1 or from a variant thereof havingincreasingly preferred 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 additionalpoint mutations to give those mutations resulting in any of the aminoacid substitutions according to the invention as defined above, i.e. inaddition to those mutations by which at least one asparagine (N) orglutamine (Q) residue on the surface of said molecule has been replacedwith A, C, F, G, H, I, K, L, M, P, R, S, T, V, W or Y. Preferably inaddition to those, wherein N has been replaced with A, G, K, R, S or T,preferably with S or T, or in addition to those, wherein Q has beenreplaced with A, G, I, K, R, S or T, preferably with S or T.

This is because, as explained above, various point mutations can beintroduced into α-amylase molecules, which can exert advantageousactions in principle independently of one another. Thus it is possibleto carry out substitutions according to the invention on those moleculeswhich have been improved in particular with respect to specialperformance aspects, stability or, for example, allergenicity. Theyinclude in particular also non-inventive point mutations. This approachof generating in the end amino acid substitutions according to theinvention is based on the corresponding nucleic acids, and the order inwhich these various mutations are carried out is in principletechnically irrelevant here.

The present invention further relates to vectors which comprise apreviously specified nucleic acid of the invention.

This is because, in order to handle the nucleic acids relevant to theinvention and to carry out in particular the methods of the inventiontherewith and to prepare the production of proteins of the invention,they are suitably ligated into vectors. Such vectors and thecorresponding methods are described in detail in the prior art. A largenumber and variety of vectors are commercially available, both forcloning and for expression. These include, for example, vectors derivedfrom bacterial plasmids, from bacteriophages or from viruses, or mainlysynthetic vectors. They are further distinguished by the nature of thecell types in which they are able to establish themselves, for exampleaccording to vectors for Gram-negative, Gram-positive bacteria, foryeasts or for higher eukaryotes. They are suitable starting points, forexample for molecular-biological and biochemical studies, formutagenesis of the nucleic acid sections present therein and forexpression of the gene in question or the corresponding protein. Theyare virtually indispensable, in particular for preparing constructs fordeleting or enhancing expression, as is revealed by the relevant priorart.

Vectors are particularly suitable for making sequence variationsaccording to the invention, for example via site-directed mutagenesis,as illustrated in Example 3.

In one embodiment, vectors according to the invention are cloningvectors.

This is because cloning vectors are suitable for molecular-biologicalcharacterization of the gene of interest, in addition to its storage,biological amplification or selection. At the same time, they aretransportable and storable forms of the claimed nucleic acids and arealso starting points for molecular-biological techniques not associatedwith cells, such as, for example, PCR or in vitro mutagenesis methods.

It is possible, for example, to convert a cloning vector carrying thegene for an α-amylase described in the prior art to a cloning vector ofthe invention by carrying out a mutation according to the invention.

Vectors according to the invention are preferably expression vectors.

This is because such expression vectors are the basis of establishingthe corresponding nucleic acids in biological production systems andthereby producing the corresponding proteins, since they enable the geneproduct in question to be transcribed and translated, i.e. synthesized,in vivo. Preferred embodiments of this subject matter of the inventionare expression vectors that carry the genetic elements required forexpression, for example the natural promoter originally located upstreamof said gene or a promoter from a different organism. These elements maybe arranged, for example, in the form of an “expression cassette”.Alternatively, individual or all regulatory elements may also beprovided by the particular host cell. Particular preference is given toexpression vectors tuned to the chosen expression system, in particularthe host cell (see below), with respect to further properties, such as,for example, optimal copy number.

Providing an expression vector is usually the best option for amplifyinga protein according to the invention, preferably in combination withcoacting proteins according to the invention, and thus increasing theactivity in question or making it available to quantitative preparation.

A separate subject matter of the invention are cells which, aftergenetic modification, comprise any of the previously specified nucleicacids of the invention.

This is because said cells contain genetic information for the synthesisof a protein according to the invention and are used in the method ofthe invention, if the α-amylases in question are obtainedbiotechnologically. Said cells are understood as meaning in particularthose cells which have been provided with the nucleic acids of theinvention by methods known per se or which derived from such cells. Thisinvolves selecting suitably those host cells which can be cultured in acomparatively simple manner and/or deliver high yields of product.

They enable, for example, the corresponding genes to be amplified, orelse mutagenesis thereof, if an in vivo metagenesis method is carriedout, or transcription and translation, and ultimately biotechnologicalproduction of the proteins in question. Said genetic information may bepresent either extrachromosomally as a separate genetic element, i.e.located on a plasmid in the case of bacteria, or integrated in achromosome. The choice of a suitable system depends on problems such as,for example, type and duration of storage of said gene, or of theorganism or the type of mutagenesis or selection. Such possibleimplementations are known to the molecular biologist.

This also explains the preferred embodiment, according to which saidnucleic acid in such a cell is part of a vector, in particular part ofan above-described vector of the invention.

This is because the above-described advantages in handling, storage,expression, etc., of the nucleic acid in question are associatedtherewith.

Among these cells, preference is in each case given to a host cell thatis a bacterium, in particular one that secretes the α-amylase variantformed into the surrounding medium.

This is because bacteria are characterized by short generation times andlow demands on culturing conditions. This enables inexpensive methods tobe established. Moreover, there is plenty of variable experienceregarding bacteria in fermentation technology. Gram-negative orGram-positive bacteria may be suitable for a special production for verydifferent reasons which may be determined experimentally in theindividual case, such as nutrient sources, rate of product formation,time required, etc.

An additional advantageous embodiment relates to the fact that mostindustrially important α-amylases have originally been found as enzymessecreted by the relevant microorganisms, in particular bacteria, intothe surrounding medium. This is because they are usually digestiveenzymes in vivo. Accordingly, it is advantageous if the industriallyproduced enzymes of the invention are likewise secreted into thesurrounding medium, since they can be worked up therefrom without celldisruption and therefore comparatively easily. This may be achieved, forexample, by adding appropriate sequences to the genes in question—ifthese are not present anyway—that code for a leader peptide which causesthe cell in question to export them. An alternative in Gram-negativebacteria, for example, consists of partially opening the outer membraneto release proteins, as described, for example, in the application WO01/81597 A1.

In a preferred embodiment, the bacterium is a Gram-negative bacterium,in particular one of the genera Escherichia coli, Klebsiella,Pseudomonas or Xanthomonas, in particular E. coli K12, E. coli B orKlebsiella planticola strains, and very particular derivatives of thestrains Escherichia coli BL21 (DE3), E. coli RV308, E. coli DH5α, E.coli JM109, E. coli XL-1 or Klebsiella planticola (Rf).

This is because these species and strains are established in the priorart for molecular-biological procedures and biotechnological productionprocesses. They are moreover available via generally accessible straincollections such as the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Mascheroder Web 1b, 38124 Braunschweig, Germany(http://www.dsmz.de) or from commercial sources. In addition, they canbe optimized for specific production conditions in combination withother components such as, for example, vectors, which are likewiseavailable in large numbers.

Gram-negative bacteria such as, for example, E. coli secrete amultiplicity of proteins into the periplasmic space. This may beadvantageous for special applications. There are, as mentioned, alsoprocesses known, by which Gram-negative bacteria too export theexpressed proteins.

In an alternative embodiment which is no less preferred, the bacteriumis a Gram-positive bacterium, in particular one of the genera Bacillus,Staphylococcus or Corynebacterium, very particularly of the speciesBacillus lentus, B. licheniformis, B. amyloliquefaciens, B. subtilis, B.globigii or B. alcalophilus, Staphylococcus carnosus or Corynebacteriumglutamicum, and among these in turn, very particularly preferably a B.licheniformis DSM 13 derivative.

This is because Gram-positive bacteria are fundamentally different fromGram-negative ones in that secreted proteins are immediately releasedinto the nutrient medium surrounding the cells, from which medium theexpressed proteins according to the invention can be purified directly,if desired. Moreover, they are related to most source organisms forindustrially important enzymes or are identical thereto and usuallyproduce themselves comparable enzymes so that they have a similar codonusage and their protein synthesis apparatus is by nature geared theretoaccordingly. B. licheniformis DSM 13 which characterizes a veryparticularly preferred embodiment is likewise a very commonbiotechnological producer strain in the prior art.

Another embodiment of the present invention consists of methods ofproducing an above-described α-amylase variant.

The, in a narrower sense inventive, methods of increasing the stabilityof an α-amylase to solvent-exerted hydrolysis have already beendescribed above. They refer primarily to molecular-biological steps inorder to generate such variants in the first place. The methods definedat this point, which are in a wider sense likewise inventive, are thosewhich are industrially required in order to produce quantitative amountsof the α-amylase variants of the invention. Thus they primarily refer tobiotechnological methods of producing variants according to theinvention—apart from the rather only theoretically relevant chemicalsynthesis of said enzymes. Said methods usually involve microorganismstrains which, by applying molecular-biological techniques known per se,have been enabled to produce α-amylase variants which in the course ofthe present invention have been acknowledged as being advantageousaccording to the invention.

The method of the invention, which has been illustrated and which isbased in particular on mutagenesis, may thus be introduced as acharacterizing section into an established biotechnological method ofproducing α-amylases, i.e. may be combined with such a method.

The formation of a protein in question in a strain employed forproduction is detected by way of enzymic detection of the enzymeactivities in question by detection reactions known per se for α-amylaseactivity. For detection at the molecular-biological level, the proteinsdepicted in the present sequence listing can be synthesized by customarymethods and antibodies can be raised thereto. These proteins can then bedetected, for example, via appropriate Western blots. Said antibodiesreact particularly preferably to those surface regions which have beenmodified according to the invention, since the latter can bedistinguished from the non-inventive variants.

The aim of these methods is to deliver the α-amylases in question totheir particular applications. To this end, any work-up, purificationand formulation steps established in biotechnology can be used. Theseinclude, for example, the method of refining concentrated enzymesolutions, described in the application WO 2004/067557 A1, which ischromatography-based and which consequently can also be appliedsuccessfully to amylase preparations. Said method is the basis of theapplication DE 10360841.9 which has not been prepublished and accordingto which the solutions in question which have been obtained inter aliaby a chromatographic step can be processed further to give enzymegranules. Further method aspects which may likewise be combinedadvantageously with the present invention are revealed in theapplications DE 102004021384.4 and DE 192004019528 which have likewisenot been prepublished and according to which the storage stability andabrasion resistance of such granules can be increased by incorporatingglycerol or 1,2-propanediol or by a special coating.

Said methods, in particular their component steps, in which the enzymepreparation is still in a liquid form, are improved according to theinvention by the fact that the present α-amylase variant according tothe invention is less prone to denaturation, resulting in a smaller lossof total activity. This enables, for example, α-amylase granules to beproduced which contain a higher proportion of active substances.

The production methods are preferably methods which are carried outusing an above-specified nucleic acid of the invention, preferably usinga previously specified vector of the invention and particularlypreferably using a previously specified cell of the invention.

This is because said nucleic acids, in particular the nucleic acidsspecified in the sequence listing, make available the correspondinglypreferred genetic information in a microbiologically usable form, i.e.genetic engineering production processes. Increasing preference is givento providing said nucleic acids on a vector which can be utilizedparticularly successfully by the host cell, or such cells themselves.The production processes in question are known per se to the skilledworker.

Embodiments of the present invention may also be, on the basis of thecorresponding nucleic acid sequences, cell-free expression systems whichcarry out protein biosynthesis in vitro. All elements alreadyillustrated above may also be combined to give new methods for producingproteins according to the invention. In this connection, a multiplicityof possible combinations of method steps is conceivable for each proteinaccording to the invention and, as a result, optimal methods must bedetermined experimentally for each specific individual case.

Further preference is given to those methods in which one, preferablytwo or more and particularly preferably all codons of the nucleotidesequence have been adapted to the codon usage of the host strain. Thisis because the transfer of any of said genes to a less related speciesmay be utilized particularly successfully for the synthesis of theprotein in question, if their codon usage has been optimizedaccordingly.

Agents containing an above-described α-amylase variant of the inventionconstitute a separate subject-matter of the invention.

This means any agent in which the α-amylase in question is applied inany form, i.e. brought into the desired hydrolytic contact with itssubstrate, starch or starch-like polysaccharide, or is preparedtherefor. The desired contact usually takes place in an aqueousenvironment which is advantageously buffered to an appropriate pH and,where appropriate, contains further beneficial factors. These include,for example, further enzymes which further convert the immediatereaction products, for example with regard to starch liquefaction forthe production of food or animal feed or for ethanol production. Alsoincluded here are low-molecular weight compounds which are included, forexample, in nascent oligosaccharides, such as cyclodextrins, orlow-molecular compounds which further solubilize the cleavage productsor have a beneficial effect on the overall process, such as, forexample, surfactants within the framework of a detergent formulation.They may also be agents in which the desired analytical activity is tobe induced only with a large time delay, such as, for example, intemporary bonding processes, according to which the α-amylase variant isadded early to the adhesive in question but becomes actually active onlyafter a long time, due to an increase in the water content. Thisapplies, for example, also to detergents and cleansers which areintended to act on the substrate at the moment of dilution in the washliquor, only after a storage phase.

One embodiment of this subject matter of the invention are those agentswhich are a detergent or cleanser.

The properties and important ingredients according to the invention ofsuch detergents and cleansers are illustrated in more detailhereinbelow. At this point, there will be first an overview of the mostimportant embodiments, which are therefore particularly preferredaccording to the invention, of such detergents and cleansers:

A detergent or cleanser according to the invention, comprising from0.000001 percent by weight to 5% by weight, in particular from 0.00001to 3% by weight, of the α-amylase variant;

a detergent or cleanser according to the invention, which additionallyincludes other enzymes, in particular hydrolytic enzymes oroxidoreductases, particularly preferably further amylases, proteases,lipases, cutinases, hemicellulases, cellulases, β-glucanases, oxidases,peroxidases, perhydrolases and/or laccases;

a detergent or cleanser according to the invention, wherein theα-amylase variant is stabilized and/or its contribution to the washingor cleaning performance of the agent is increased by any of the othercomponents of said agent;

a detergent or cleanser according to the invention, which is overallsolid, preferably after a compacting step for at least one of theincluded components, particularly preferably that it is overallcompacted;

a detergent or cleanser according to the invention, which is overallliquid, gel-like or paste-like, preferably with encapsulation of atleast one of the included components, particularly preferably withencapsulation of at least one of the included enzymes, very particularlypreferably with encapsulation of the α-amylase variant.

One important field of use of amylases is that as active components indetergents or cleansers for cleaning textiles or solid surfaces, suchas, for example, crockery, floors or utensils. In these applications,the amylolytic activity serves to break down by hydrolysis, or detachfrom the substrate, carbohydrate-containing contaminations and inparticular those based on starch. In this connection, they may be usedalone, in suitable media or else in detergents or cleansers. Theconditions to be chosen for this, such as, for example, temperature, pH,ionic strength, redox conditions or mechanical effects, should beoptimized for the particular cleaning problem, i.e. in relation to thesoiling and the substrate. Thus, usual temperatures for detergents andcleansers are in ranges from 10° C. for manual compositions via 40° C.and 60° C. up to 950 for machine compositions or for industrialapplications. Since the temperature can usually be adjusted continuouslyin modern washing and dishwashing machines, all intermediatetemperatures are also included. The ingredients of the relevant agentsare preferably also matched to one another. The other conditions canlikewise be designed very specifically for the particular cleaningpurpose via the other components of said agents, illustrated below.

Preferred detergents and cleansers of the invention are distinguished bythe washing or cleaning performance of the agent in question beingimproved by adding an α-amylase variant of the invention, compared withthe formulation without this variant, under any of the conditionsdefinable in this way. Preference is given to synergies with respect tocleaning performance.

An α-amylase variant of the invention can be used both in compositionsfor large-scale consumers or industrial users and in products for theprivate consumer, and all presentations which are expedient and/orestablished in the art also represent embodiments of the presentinvention. The detergents or cleansers of the invention thus mean anyconceivable types of cleaning compositions, both concentrates andcompositions to be applied in an undiluted form; for use on a commercialscale, in the washing machine or for washing or cleaning by hand. Theyinclude, for example, detergents for textiles, carpets or naturalfibers, for which agents the term detergent is used according to thepresent invention. They include also, for example, dishwashing agentsfor dishwashers or manual washing-up liquids or cleaners for hardsurfaces such as metal, glass, porcelain, ceramics, tiles, stone,painted surfaces, plastics, wood or leather; for these, the termcleanser is used according to the present invention. Any type ofdetergent or cleanser is an embodiment of the present invention, if anamylase of the invention has been added to it.

Embodiments of the present invention comprise any presentationsestablished in the art and/or any expedient presentations of thecompositions of the invention. These include, for example, solids,pulverulent, liquid, gel-like or paste-like compositions, whereappropriate also composed of two or more phases, compressed oruncompressed; they also include for example: extrudates, granules,tablets or pouches, packaged both in large containers and in portions.

α-Amylases are combined in agents of the invention, for example, withone or more of the following ingredients: nonionic, anionic and/orcationic surfactants, bleaches, bleach activators, bleach catalysts,builders and/or cobuilders, solvents, thickeners, sequestering agents,electrolytes, optical brighteners, antiredeposition agents, corrosioninhibitors, in particular silver protectants, soil release agents, colortransfer inhibitors, foam inhibitors, abrasives, dyes, fragrances,antimicrobial agents, UV stabilizers, enzymes such as, for example,proteases, (where appropriate other) amylases, lipases, cellulases,hemicellulases or oxidases, stabilizers, in particular enzymestabilizers, and other components known in the art.

The nonionic surfactants used are preferably alkoxylated, advantageouslyethoxylated, in particular primary alcohols having preferably from 8 to18 carbon atoms and, on average, from 1 to 12 mol of ethylene oxide (EO)per mole of alcohol, in which the alcohol radical can be linear or,preferably, methyl-branched in the 2-position or can comprise linear andmethyl-branched radicals in a mixture as are customarily present in oxoalcohol radicals. Particular preference is, however, given to alcoholethoxylates containing linear radicals of alcohols of native originhaving from 12 to 18 carbon atoms, for example from coconut, palm,tallow fatty or oleyl alcohol, and, on average, from 2 to 8 EO per moleof alcohol. Preferred ethoxylated alcohols include, for example,C₁₂₋₁₄-alcohols having 3 EO or 4 EO, C₉₋₁₁-alcohol having 7 EO,C₁₃₋₁₅-alcohols having 3 EO, 5 EO, 7 EO or 8 EO, C₁₂₋₁₈-alcohols having3 EO, 5 EO or 7 EO, and mixtures of these, such as mixtures ofC₁₂₋₁₄-alcohol having 3 EO and C₁₂₋₁₈-alcohol having 5 EO. The degreesof ethoxylation given are statistical averages which may be an integeror a fraction for a specific product. Preferred alcohol ethoxylates havea narrowed homolog distribution (narrow range ethoxylates, NRE). Inaddition to these nonionic surfactants, fatty alcohols having more than12 EO can also be used. Examples thereof are tallow fatty alcohol having14 EO, 25 EO, 30 EO or 40 EO.

A further class of preferably used nonionic surfactants which are usedeither as the sole nonionic surfactant or in combination with othernonionic surfactants are alkoxylated preferably ethoxylated orethoxylated and propoxylated fatty acid alkyl esters, preferably havingfrom 1 to 4 carbon atoms in the alkyl chain, in particular fatty acidmethyl esters.

The application DE 1020040430.6 which has not been published previouslyreveals cleansers, in particular machine dishwashing agents, which (a)comprise one or more nonionic surfactants having the following generalformula:

where R¹ is a C₆₋₂₄-alkyl or -alkenyl radical, the groups R² and R³ arein each case particular hydrocarbon radicals and the indices w, x, y, zare in each case integers up to 6, or a surfactant system of at leastone nonionic surfactant F of the general formula:R¹—CH(OH)CH₂O-(AO)_(w)-(A′O)_(x)-(A″O)_(y)-(A′″O)_(z)—R²and at least one nonionic surfactant G of the general formula:R¹—O-(AO)_(w)-(A′O)_(x)-(A″O)_(y)-(A′″O)_(z)—R²where in each case R¹ is a C₆₋₂₄-alkyl or -alkenyl radical, R² is ahydrocarbon radical having from 2 to 26 carbon atoms, A, A′, A″ and A′″are in each case particular hydrocarbon radicals, and w, x, y and z arein each case values of up to 25, said surfactant system having thesurfactants F and G in a weight ratio of between 1:4 and 100:1, and aspecial α-amylase variant as component (b).

These surfactants can be combined, in particular in machine dishwashingagents, also with α-amylases which correspond to the present invention,in particular if they have, besides the substitutions according to theinvention, those which can be found in one or more of the applicationsWO 96/23873 A1, WO 00/60060 A2 and WO 01/66712 A2. This applies inparticular to the case in which the commercial product Stainzyme® fromNovozymes, which falls under these applications, is improved in furtherpositions and is additionally provided with at least one substitution ofthe invention, since in principle an additive effect of the variousmodifications must be assumed.

A further class of nonionic surfactants which can advantageously be usedare the alkyl polyglycosides (APG). Alkyl polyglycosides which may beused satisfy the general formula RO(G)_(z), in which R is a linear orbranched, in particular methyl-branched in the 2-position, saturated orunsaturated, aliphatic radical having from 8 to 22, preferably from 12to 18 carbon atoms, and G is the symbol which stands for a glycose unithaving 5 or 6 carbon atoms, preferably for glucose. The degree ofglycosylation z is here between 1.0 and 4.0, preferably between 1.0 and2.0 and in particular between 1.1 and 1.4. Preference is given to usinglinear alkyl polyglucosides, i.e. alkyl polyglycosides in which thepolyglycosyl radical is a glucose radical, and the alkyl radical is ann-alkyl radical.

Nonionic surfactants of the amine oxide type, for example N-cocoalkyl-N,N-dimethylamine oxide and N-tallow alkyl-N, N-dihydroxyethylamine oxide,and of the fatty acid alkanolamides may also be suitable. The proportionof these nonionic surfactants is preferably no more than that of theethoxylated fatty alcohols, in particular no more than half thereof.

Further suitable surfactants are polyhydroxy fatty acid amides of theformula (II)

in which RCO is an aliphatic acyl radical having from 6 to 22 carbonatoms, R¹ is hydrogen, an alkyl or hydroxyalkyl radical having from 1 to4 carbon atoms and [Z] is a linear or branched polyhydroxyalkyl radicalhaving from 3 to 10 carbon atoms and from 3 to 10 hydroxyl groups. Thepolyhydroxy fatty acid amides are known substances which can usually beobtained by reductive amination of a reducing sugar with ammonia, analkylamine or an alkanolamine and subsequent acylation with a fattyacid, a fatty acid alkyl ester or a fatty acid chloride.

The group of polyhydroxy fatty acid amides also includes compounds ofthe formula (III)

in which R is a linear or branched alkyl or alkenyl radical having from7 to 12 carbon atoms, R¹ is a linear, branched or cyclic alkyl radicalor an aryl radical having from 2 to 8 carbon atoms, and R² is a linear,branched or cyclic alkyl radical or an aryl radical or an oxy-alkylradical having from 1 to 8 carbon atoms, where C₁₋₄-alkyl or phenylradicals are preferred, and [Z] is a linear polyhydroxyalkyl radicalwhose alkyl chain is substituted with at least two hydroxyl groups, oralkoxylated, preferably ethoxylated or propoxylated, derivatives of thisradical.

[Z] is preferably obtained by reductive amination of a reducing sugar,for example glucose, fructose, maltose, lactose, galactose, mannose orxylose. The N-alkoxy- or N-aryloxy-substituted compounds may beconverted, for example by reaction with fatty acid methyl esters in thepresence of an alkoxide as catalyst, into the desired polyhydroxy fattyacid amides.

The anionic surfactants used are, for example, those of the sulfonateand sulfate type. Suitable surfactants of the sulfonate type arepreferably C₉₋₁₃-alkylbenzene sulfonates, olefin sulfonates, i.e.mixtures of alkene and hydroxyalkane sulfonates, and disulfonates, asobtained, for example, from C₁₂₋₁₈-monoolefins having a terminal orinternal double bond by sulfonation with gaseous sulfur trioxide andsubsequent alkaline or acidic hydrolysis of the sulfonation products.Also suitable are alkane sulfonates which are obtained fromC₁₂₋₁₈-alkanes, for example, by sulfochlorination or sulfoxidation withsubsequent hydrolysis or neutralization. Likewise suitable are also theesters of α-sulfo fatty acids (estersulfonates), for example theα-sulfonated methyl esters of hydrogenated coconut, palm kernel ortallow fatty acids.

Further suitable anionic surfactants are sulfated fatty acid glycerolesters. Fatty acid glycerol esters mean the mono-, di- and triesters,and mixtures thereof, as are obtained during the preparation byesterification of a monoglycerol with from 1 to 3 mol of fatty acid orduring the transesterification of triglycerides with from 0.3 to 2 molof glycerol. Preferred sulfated fatty acid glycerol esters are here thesulfation products of saturated fatty acids having from 6 to 22 carbonatoms, for example of caproic acid, caprylic acid, capric acid, myristicacid, lauric acid, palmitic acid, stearic acid or behenic acid.

Preferred alk(en)yl sulfates are the alkali metal, and in particular thesodium, salts of sulfuric half-esters of C₁₂-C₁₈-fatty alcohols, forexample of coconut fatty alcohol, tallow fatty alcohol, lauryl,myristyl, cetyl or stearyl alcohol or of C₁₀-C₂₀-oxo alcohols and thosehalf-esters of secondary alcohols of these chain lengths. Furtherpreferred are alk(en)yl sulfates of the said chain length which comprisea synthetic, petrochemical-based straight-chain alkyl radical which haveanalogous degradation behaviour to the equivalent compounds based onfatty chemical raw materials. From a washing performance viewpoint,preference is given to C₁₂-C₁₆-alkyl sulfates and C₁₂-C₁₅-alkylsulfates, and C₁₄-C₁₅-alkyl sulfates. 2,3-Alkyl sulfates are alsosuitable anionic surfactants.

The sulfuric monoesters of straight-chain or branched C₇₋₂₁-alcoholsethoxylated with from 1 to 6 mol of ethylene oxide, such as2-methyl-branched C₉₋₁₁-alcohols having, on average, 3.5 mol of ethyleneoxide (EO) or C₁₂₋₁₈-fatty alcohols having from 1 to 4 EO, are alsosuitable. Owing to their high foaming behaviour, they are used incleaning agents only in relatively small amounts, for example in amountsup to 5% by weight, usually from 1 to 5% by weight.

Further suitable anionic surfactants are also the salts ofalkylsulfosuccinic acid, which are also referred to as sulfosuccinatesor as sulfosuccinic esters and which are monoesters and/or diesters ofsulfosuccinic acid with alcohols, preferably fatty alcohols and, inparticular, ethoxylated fatty alcohols. Preferred sulfosuccinatescontain C₈₋₁₈-fatty alcohol radicals or mixtures thereof. Particularlypreferred sulfosuccinates contain a fatty alcohol radical derived fromethoxylated fatty alcohols, which are themselves nonionic surfactants(see below for description). In this connection, sulfosuccinates whosefatty alcohol radicals are derived from ethoxylated fatty alcoholshaving a narrowed homologue distribution are, in turn, particularlypreferred. Likewise, it is also possible to use alk(en)ylsuccinic acidhaving preferably from 8 to 18 carbon atoms in the alk(en)yl chain orsalts thereof.

Further suitable anionic surfactants are, in particular, soaps.Saturated fatty acid soaps such as the salts of lauric acid, myristicacid, palmitic acid, stearic acid, hydrogenated erucic acid and behenicacid, and, in particular, soap mixtures derived from natural fattyacids, for example coconut, palm kernel or tallow fatty acids, aresuitable.

The anionic surfactants including soaps may be present in the form oftheir sodium, potassium or ammonium salts, and as soluble salts oforganic bases such as mono-, di- or triethanolamine. The anionicsurfactants are preferably in the form of their sodium or potassiumsalts, in particular in the form of the sodium salts.

The surfactants may be present in the cleaning agents or washing agentsaccording to the invention in an overall amount of from preferably 5% byweight to 50% by weight, in particular from 8% by weight to 30% byweight, based on the finished agent.

Agents according to the invention may contain bleaches. Of the compoundswhich serve as bleaches and produce H₂O₂ in water, sodium percarbonate,sodium perborate tetrahydrate and sodium perborate monohydrate are ofparticular importance. Other bleaches which can be used are, forexample, peroxopyrophosphates, citrate perhydrates and H₂O₂-producingperacidic salts or peracids, such as persulfates or persulfuric acid.Also useful is the urea peroxohydrate percarbamide which can bedescribed by the formula H₂N—CO—NH₂.H₂O₂. In particular when used forcleaning hard surfaces, for example for machine dishwashing, the agents,if desired, may also contain bleaches from the group of organicbleaches, although the use thereof is possible in principle also inagents for washing textiles. Typical organic bleaches are diacylperoxides such as, for example, dibenzoyl peroxide. Further typicalorganic bleaches are the peroxy acids, specific examples being alkylperoxy acids and aryl peroxy acids. Preferred representatives are peroxybenzoic acid and its ring-substituted derivatives, such asalkylperoxybenzoic acids, but also peroxy-α-naphthoic acid and magnesiummonoperphthalate, the aliphatic or substituted aliphatic peroxy acidssuch as peroxylauric acid, peroxystearic acid,ε-phthalimidoperoxycaproic acid (phthalimidoperoxyhexanoic acid, PAP),o-carboxybenzamidoperoxycaproic acid, N-nonenylamidoperadipic acid andN-nonenylamidopersuccinate, and aliphatic and araliphaticperoxydicarboxylic acids such as 1,12-diperoxycarboxylic acid,1,9-diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassylic acid,diperoxyphthalic acids, 2-decyldiperoxybutane-1,4-dioic acid, N,N-terephthaloyl-di(6-aminopercaproic acid) may be used.

The bleach content of the agents may be from 1 to 40% by weight and, inparticular, from 10 to 20% by weight, using advantageously perboratemonohydrate or percarbonate. The applications WO 99/63036 and WO99/63037, respectively, disclose a synergistic use of amylase withpercarbonate and of amylase with percarboxylic acid.

In order to achieve improved bleaching action in cases of washing attemperatures of 60° C. and below, and in particular in the case oflaundry pretreatment, the agents may also include bleach activators.Bleach activators which can be used are compounds which, underperhydrolysis conditions, give aliphatic peroxocarboxylic acids havingpreferably from 1 to 10 carbon atoms, in particular from 2 to 4 carbonatoms, and/or substituted or unsubstituted perbenzoic acid. Substanceswhich carry O- and/or N-acyl groups of the said number of carbon atomsand/or substituted or unsubstituted benzoyl groups are suitable.Preference is given to plurally acylated alkylenediamines, in particulartetraacetylethylenediamine (TAED), acylated triazine derivatives, inparticular 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT),acylated glycoluriles, in particular 1,3,4,6-tetraacetylglycolurile(TAGU), N-acylimides, in particular N-nonanoylsuccinimide (NOSI),acylated phenol sulfonates, in particular n-nonanoyl- orisononanoyloxybenzene sulfonate (n- or iso-NOBS), acylatedhydroxycarboxylic acids such as triethyl-O-acetyl citrate (TEOC),carboxylic anhydrides, in particular phthalic anhydride, isatoicanhydride and/or succinic anhydride, carboxamides such asN-methyldiacetamide, glycolide, acylated polyhydric alcohols, inparticular triacetin, ethylene glycol diacetate, isopropenyl acetate,2,5-diacetoxy-2,5-dihydrofuran and the enol esters disclosed in Germanpatent applications DE 196 16 693 and DE 196 16 767, and acetylatedsorbitol and mannitol, or mixtures thereof described in European patentapplication EP 0 525 239 (SORMAN), acylated sugar derivatives, inparticular pentaacetylglucose (PAG), pentaacetylfructose,tetraacetylxylose and octaacetyllactose, and acetylated, optionallyN-alkylated glucamine or gluconolactone, triazole or triazolederivatives and/or particulate caprolactams and/or caprolactamderivatives, preferably N-acylated lactams, for exampleN-benzoylcaprolactam and N-acetylcaprolactam. Hydrophilicallysubstituted acyl acetals and acyl lactams are likewise used withpreference. It is also possible to use combinations of conventionalbleach activators. Nitrile derivatives such as cyanopyridines, nitrilequats, e.g. N-alkylammoniumacetonitriles, and/or cyanamide derivativesmay also be used. Preferred bleach activators are sodium4-(octanoyloxy)benzenesulfonate, n-nonanoyl- orisononanoyloxybenzenesulfonate (n- or iso-NOBS),undecenoyloxybenzenesulfonate (UDOBS), sodiumdodecanoyloxybenzenesulfonate (DOBS), decanoyloxybenzoic acid (DOBA, OBC10) and/or dodecanoyloxybenzenesulfonate (OBS 12), andN-methylmorpholinium acetonitrile (MMA). Such bleach activators may bepresent in the customary quantitative range from 0.01 to 20% by weight,preferably in amounts from 0.1 to 15% by weight, in particular 1% byweight to 10% by weight, based on the total composition.

In addition to the conventional bleach activators or instead of them, itis also possible for “bleach catalysts” to be present. These substancesare bleach-enhancing transition metal salts or transition metalcomplexes such as, for example, Mn, Fe, Co, Ru or Mo saline complexes orcarbonyl complexes. Mn, Fe, Co, Ru, Mo, Ti, V and Cu complexescontaining N-containing tripod ligands, and Co, Fe, Cu and Ru aminecomplexes are also suitable as bleach catalysts. Acetonitrilederivatives, according to WO 99/63038, and bleach-activating transitionmetal complex compounds, according to WO 99/63041 are capable ofdeveloping a bleach-activating action in combination with amylases.

Agents according to the invention usually contain one or more builders,in particular zeolites, silicates, carbonates, organic cobuilders and,where no ecological reasons oppose their use, also phosphates. Thelatter are the preferred builders for use in particular in cleaningagents for machine dishwashing.

Compounds which may be mentioned here are crystalline, layered sodiumsilicates of the general formula NaMSi_(x)O_(2x-1).yH₂O, where M issodium or hydrogen, x is a number from 1.6 to 4, preferably from 1.9 to4.0, and y is a number from 0 to 20, and preferred values for x are 2, 3or 4. Preferred crystalline phyllosilicates of the formula indicated arethose where M is sodium and x adopts the values 2 or 3. In particular,both β- and δ-sodium disilicates Na₂Si₂O₅.yH₂O are preferred. Compoundsof this kind are sold, for example, under the name SKS® (Clariant).Thus, SKS-6° is primarily a δ-sodium disilicate having the formulaNa₂Si₂O₅.yH₂O, and SKS-7® is primarily the β-sodium disilicate. Reactingthe δ-sodium disilicate with acids (for example citric acid or carbonicacid) gives kanemite NaHSi₂O₅.yH₂O, sold under the names SKS-9® and,respectively, SKS-10® (Clariant). It may also be advantageous to usechemical modifications of the said phyllosilicates. The alkalinity ofthe phyllosilicates, for example, can thus be suitably influenced.Phyllosilicates doped with phosphate or with carbonate have, compared tothe δ-sodium disilicate, altered crystal morphologies, dissolve morerapidly and display an increased calcium binding ability, compared toδ-sodium disilicate. Phyllosilicates of the general empirical formulaxNa₂O.ySiO₂.zP₂O₅ where the x-to-y ratio corresponds to a number from0.35 to 0.6, the x-to-z ratio to a number from 1.75 to 1200 and they-to-z ratio to a number from 4 to 2800 are to be mentioned inparticular. The solubility of the phyllosilicates may also be increasedby using particularly finely granulated phyllosilicates. It is alsopossible to use compounds of the crystalline phyllosilicates with otheringredients. Compounds which may be mentioned here are in particularthose with cellulose derivatives which have advantageous disintegratingaction and are used in particular in washing agent tablets, and thosewith polycarboxylates, for example citric acid, or polymericpolycarboxylates, for example copolymers of acrylic acid.

It is also possible to use amorphous sodium silicates having a Na₂O:SiO₂modulus of from 1:2 to 1:3.3, preferably from 1:2 to 1:2.8, and inparticular from 1:2 to 1:2.6, which have delayed dissolution andsecondary washing properties. The dissolution delay relative toconventional amorphous sodium silicates can have been induced by variousmeans, for example by surface treatment, compounding,compaction/compression or by overdrying. Within the scope of thisinvention, the term “amorphous” also means “X-ray amorphous”. This meansthat in X-ray diffraction experiments the silicates do not give thesharp X-ray refractions typical of crystalline substances, but instead,at best, one or more maxima of these scattered X-rays, which have awidth of several degree units of the diffraction angle. However,particularly good builder properties will very likely result if, inelectron diffraction experiments, the silicate particles give poorlydefined or even sharp diffraction maxima. This is to be interpreted tothe effect that the products have microcrystalline regions with a sizefrom 10 to a few hundred nm, preference being given to values up to atmost 50 nm and in particular up to at most 20 nm. Particular preferenceis given to compressed/compacted amorphous silicates, compoundedamorphous silicates and overdried X-ray amorphous silicates.

A finely crystalline, synthetic zeolite containing bonded water, whichmay be used where appropriate, is preferably zeolite A and/or P. Aszeolite P, zeolite MAP® (commercial product from Crosfield) isparticularly preferred. However, zeolite X and mixtures of A, X and/or Pare also suitable. A product which is commercially available and can beused with preference within the scope of the present invention is, forexample, also a co-crystallizate of zeolite X and zeolite A (approx. 80%by weight zeolite X), which is sold by CONDEA Augusta S.p.A. under thetrade name VEGOBOND AX® and can be described by the formulanNa₂O.(1-n)K₂O.Al₂O₃.(2-2.5)SiO₂.(3.5-5.5)H₂OSuitable zeolites have an average particle size of less than 10 μm(volume distribution; measurement method: Coulter counter) andpreferably contain from 18 to 22% by weight, in particular from 20 to22% by weight, of bonded water.

Use of the generally known phosphates as builder substances is of coursealso possible, provided such a use should not be avoided for ecologicalreasons. Among the multiplicity of commercially available phosphates,the alkali metal phosphates are the most important in the washing andcleaning agents industry, with pentasodium or pentapotassiumtriphosphate (sodium or potassium tripolyphosphate) being particularlypreferred.

In this connection, alkali metal phosphates is the collective term forthe alkali metal (in particular sodium and potassium) salts of thevarious phosphoric acids, it being possible to differentiate betweenmetaphosphoric acids (HPO₃)_(n) and orthophosphoric acid H₃PO₄ as wellas higher molecular weight representatives. The phosphates combineseveral advantages: they act as alkali carriers, prevent lime depositson machine parts and lime incrustations in fabrics and, moreover,contribute to the cleaning performance.

Sodium dihydrogenphosphate, NaH₂PO₄, exists as dihydrate (density 1.91gcm⁻³, melting point 60° C.) and as monohydrate (density 2.04 gcm⁻³).Both salts are white powders which are very readily soluble in water andwhich lose their water of crystallization upon heating and at 200° C.convert to the weakly acidic diphosphate (disodium hydrogendiphosphate,Na₂H₂P₂O₇), at a higher temperature to sodium trimetaphosphate (Na₃P₃O₉)and Maddrell's salt (see below). NaH₂PO₄ is acidic; it forms whenphosphoric acid is adjusted to a pH of 4.5 using sodium hydroxidesolution and the suspension is sprayed. Potassium dihydrogenphosphate(primary or monobasic potassium phosphate, potassium biphosphate, KDP),KH₂PO₄, is a white salt of density 2.33 gcm⁻³, has a melting point of2530 [decomposition with the formation of potassium polyphosphate(KPO₃)_(x)] and is readily soluble in water.

Disodium hydrogenphosphate (secondary sodium phosphate), Na₂HPO₄, is acolorless crystalline salt which is very readily soluble in water. Itexists in anhydrous form and with 2 mol (density 2.066 gcm⁻³, loss ofwater at 95° C.), 7 mol (density 1.68 gcm⁻³, melting point 48° C. withloss of 5H₂O) and 12 mol (density 1.52 gcm⁻³, melting point 35° C. withloss of 5H₂O) of water, becomes anhydrous at 100° C. and upon morevigorous heating converts to the diphosphate Na₄P₂O₇. Disodiumhydrogenphosphate is prepared by neutralizing phosphoric acid with sodasolution using phenolphthalein as indicator. Dipotassiumhydrogenphosphate (secondary or dibasic potassium phosphate), K₂HPO₄, isan amorphous, white salt which is readily soluble in water.

Trisodium phosphate, tertiary sodium phosphate, Na₃PO₄, are colorlesscrystals which, in the form of the dodecahydrate, have a density of 1.62gcm⁻³ and a melting point of 73-76° C. (decomposition), in the form ofthe decahydrate (corresponding to 19-20% P₂O₅) have a melting point of100° C. and in anhydrous form (corresponding to 39-40% P₂O₅) have adensity of 2.536 gcm⁻³. Trisodium phosphate is readily soluble in waterwith an alkaline reaction and is prepared by evaporating a solution ofexactly 1 mol of disodium phosphate and 1 mol of NaOH. Tripotassiumphosphate (tertiary or tribasic potassium phosphate), K₃PO₄, is a white,deliquescent granular powder of density 2.56 gcm⁻³, has a melting pointof 1340° C. and is readily soluble in water with an alkaline reaction.It is produced, for example, during the heating of Thomas slag withcarbon and potassium sulfate. Despite the higher price, the more readilysoluble, and therefore highly effective, potassium phosphates are oftenpreferred over corresponding sodium compounds in the cleaning agentsindustry.

Tetrasodium diphosphate (sodium pyrophosphate), Na₄P₂O₇, exists inanhydrous form (density 2.534 gcm⁻³, melting point 988° C., also 880° C.given) and as decahydrate (density 1.815-1.836 gcm⁻³, melting point 94°C. with loss of water). Both substances are colorless crystals whichdissolve in water with an alkaline reaction. Na₄P₂O₇ is formed duringthe heating of disodium phosphate to >200° C. or by reacting phosphoricacid with soda in a stoichiometric ratio and dewatering the solution byspraying. The decahydrate complexes heavy metal salts and hardnessconstituents and thus reduces the water hardness. Potassium diphosphate(potassium pyrophosphate), K₄P₂O₇, exists in the form of the trihydrateand is a colorless, hygroscopic powder of density 2.33 gcm³, which issoluble in water, the pH of the 1% strength solution at 25° C. being10.4.

Condensation of NaH₂PO₄ and KH₂PO₄ results in higher molecular weightsodium phosphates and potassium phosphates, respectively, amongst whichcyclic representatives, the sodium and potassium metaphosphates,respectively, and chain-shaped types, the sodium and potassiumpolyphosphates, respectively, can be differentiated. Particularly forthe latter, a multiplicity of names are in use: melt or thermalphosphates, Graham's salt, Kurrol's and Maddrell's salt. All highersodium and potassium phosphates are together referred to as condensedphosphates.

The industrially important pentasodium triphosphate, Na₅P₃O₁₀ (sodiumtripolyphosphate), is a nonhygroscopic, white, water-soluble salt whichis anhydrous or crystallizes with 6H₂O and is of the general formulaNaO—[P(O)(ONa)—O]_(n)—Na where n=3. In 100 g of water, about 17 g of thesalt which is free of water of crystallization dissolve at roomtemperature, approx. 20 g dissolve at 60° C., and about 32 g dissolve at100° C.; if the solution is heated at 100° C. for two hours, about 8% oforthophosphate and 15% of diphosphate form due to hydrolysis. In thepreparation of pentasodium triphosphate, phosphoric acid is reacted withsoda solution or sodium hydroxide solution in a stoichiometric ratio,and the solution is dewatered by spraying. Similarly to Graham's saltand sodium diphosphate, pentasodium triphosphate dissolves manyinsoluble metal compounds (including lime soaps, etc.). Pentapotassiumtriphosphate, K₅P₃O₁₀ (potassium tripolyphosphate), is availablecommercially, for example, in the form of a 50% strength by weightsolution (>23% P₂O₅, 25% K₂O). The potassium polyphosphates are usedwidely in the washing and cleaning agents industry. In addition, sodiumpotassium tripolyphosphates also exist which can likewise be used withinthe scope of the present invention. These form, for example, when sodiumtrimetaphosphate is hydrolysed with KOH:(NaPO₃)₃+2KOH→Na₃K₂P₃O₁₀+H₂O

According to the invention, these can be used exactly as sodiumtripolyphosphate, potassium tripolyphosphate or mixtures of these two;mixtures of sodium tripolyphosphate and sodium potassiumtripolyphosphate or mixtures of potassium tripolyphosphate and sodiumpotassium tripolyphosphate or mixtures of sodium tripolyphosphate andpotassium tripolyphosphate and sodium potassium tripolyphosphate canalso be used according to the invention.

Organic cobuilders which can be used in the washing and cleaning agentsaccording to the invention are, in particular, polycarboxylates orpolycarboxylic acids, polymeric polycarboxylates, polyaspartic acid,polyacetals, optionally oxidized dextrins, further organic cobuilders(see below), and phosphonates. These classes of substance are describedbelow.

Usable organic builder substances are, for example, the polycarboxylicacids usable in the form of their sodium salts, the term polycarboxylicacids meaning those carboxylic acids which carry more than one acidfunction. Examples of these are citric acid, adipic acid, succinic acid,glutaric acid, malic acid, tartaric acid, maleic acid, fumaric acid,sugar acids, aminocarboxylic acids, nitrilotriacetic acid (NTA), as longas such a use should not be avoided for ecological reasons, and mixturesthereof. Preferred salts are the salts of the polycarboxylic acids suchas citric acid, adipic acid, succinic acid, glutaric acid, tartaricacid, sugar acids, and mixtures thereof.

It is also possible to use the acids per se. In addition to theirbuilder action, the acids typically also have the property of anacidifying component and thus also serve to establish a lower and milderpH of washing or cleaning agents, as long as the pH resulting from themixture of the remaining components is not desired. Particular mentionshould be made here of system-compatible and environmentally safe acidssuch as citric acid, acetic acid, tartaric acid, malic acid, lacticacid, glycolic acid, succinic acid, glutaric acid, adipic acid, gluconicacid and any mixtures thereof. However, mineral acids, in particularsulfuric acid, or bases, in particular ammonium or alkali metalhydroxides, may also serve as pH regulators. The agents according to theinvention contain such regulators in amounts of preferably not more than20% by weight, in particular from 1.2% by weight to 17% by weight.

Suitable builders are also polymeric polycarboxylates; these are, forexample, the alkali metal salts of polyacrylic acid or ofpolymethacrylic acid, for example those having a relative molecular massof from 500 to 70 000 g/mol.

The molar masses given for polymeric polycarboxylates are, for thepurposes of this specification, weight-average molar masses, M_(W), ofthe respective acid form, determined in principle by means of gelpermeation chromatography (GPC), using a UV detector. The measurementwas made against an external polyacrylic acid standard which, owing toits structural similarity towards the polymers studied, providesrealistic molecular weight values. These figures differ considerablyfrom the molecular weight values obtained using polystyrenesulfonicacids as the standard. The molar masses measured againstpolystyrenesulfonic acids are usually considerably higher than the molarmasses given in this specification.

Suitable polymers are, in particular, polyacrylates which preferablyhave a molecular mass of from 2000 to 20 000 g/mol. Owing to theirsuperior solubility, preference in this group may be given in turn tothe short-chain polyacrylates which have molar masses of from 2000 to 10000 g/mol, and particularly preferably from 3000 to 5000 g/mol.

Also suitable are copolymeric polycarboxylates, in particular those ofacrylic acid with methacrylic acid and of acrylic acid or methacrylicacid with maleic acid. Copolymers which have proven to be particularlysuitable are those of acrylic acid with maleic acid which contain from50 to 90% by weight of acrylic acid and from 50 to 10% by weight ofmaleic acid. Their relative molecular mass, based on free acids, isgenerally from 2000 to 70 000 g/mol, preferably 20 000 to 50 000 g/moland in particular 30 000 to 40 000 g/mol. The (co)polymericpolycarboxylates may be used either as powders or as aqueous solution.The (co)polymeric polycarboxylates may be from 0.5 to 20% by weight, inparticular 1 to 10% by weight of the content of the agent.

To improve the solubility in water, the polymers may also containallylsulfonic acids such as, for example, allyloxybenzenesulfonic acidand methallylsulfonic acid as monomers.

Particular preference is also given to biodegradable polymers of morethan two different monomer units, for example those which contain, asmonomers, salts of acrylic acid and of maleic acid, and vinyl alcohol orvinyl alcohol derivatives, or those which contain, as monomers, salts ofacrylic acid and of 2-alkylallylsulfonic acid, and sugar derivatives.

Further preferred copolymers are those which preferably have, asmonomers, acrolein and acrylic acid/acrylic acid salts or acrolein andvinyl acetate.

Further preferred builder substances which may be mentioned are alsopolymeric aminodicarboxylic acids, their salts or their precursorsubstances. Particular preference is given to polyaspartic acids orsalts and derivatives thereof.

Further suitable builder substances are polyacetals which can beobtained by reacting dialdehydes with polyolcarboxylic acids having from5 to 7 carbon atoms and at least 3 hydroxyl groups. Preferredpolyacetals are obtained from dialdehydes such as glyoxal,glutaraldehyde, terephthalaldehyde and mixtures thereof and frompolyolcarboxylic acids such as gluconic acid and/or glucoheptonic acid.

Further suitable organic builder substances are dextrins, for exampleoligomers or polymers of carbohydrates, which can be obtained by partialhydrolysis of starches. The hydrolysis can be carried out by customaryprocesses, for example acid-catalyzed or enzyme-catalyzed processes. Thehydrolysis products preferably have average molar masses in the rangefrom 400 to 500 000 g/mol. Preference is given here to a polysaccharidehaving a dextrose equivalent (DE) in the range from 0.5 to 40, inparticular from 2 to 30, where DE is a common measure of the reducingaction of a polysaccharide compared with dextrose which has a DE of 100.It is possible to use both maltodextrins having a DE between 3 and 20and dried glucose syrups having a DE between 20 and 37, and also “yellowdextrins” and “white dextrins” with higher molar masses in the rangefrom 2000 to 30 000 g/mol.

The oxidized derivatives of such dextrins are their reaction productswith oxidation agents which are able to oxidize at least one alcoholfunction of the saccharide ring to the carboxylic acid function.Particularly preferred organic builders for agents according to theinvention are oxidized starches and derivatives thereof.

Oxydisuccinates and other derivatives of disuccinates, preferablyethylenediamine disuccinate, are also further suitable cobuilders. Here,ethylenediamine N, N′-disuccinate (EDDS) is preferably used in the formof its sodium or magnesium salts. In this connection, further preferenceis also given to glycerol disuccinates and glycerol trisuccinates.Suitable use amounts in zeolite-containing and/or silicate-containingformulations are between 3 and 15% by weight.

Further organic cobuilders which may be used are, for example,acetylated hydroxycarboxylic acids or salts thereof, which may also bepresent, where appropriate, in lactone form and which contain at least 4carbon atoms and at least one hydroxy group and at most two acid groups.

A further class of substance having cobuilder properties is thephosphonates. These are, in particular, hydroxyalkane and aminoalkanephosphonates. Among the hydroxyalkane phosphonates, 1-hydroxyethane1,1-diphosphonate (HEDP) is of particular importance as a cobuilder. Itis preferably used as sodium salt, the disodium salt being neutral andthe tetrasodium salt being alkaline (pH 9). Suitable aminoalkanephosphonates are preferably ethylenediaminetetramethylene phosphonate(EDTMP), diethylenetriaminepentamethylene phosphonate (DTPMP) and higherhomologues thereof. They are preferably used in the form of the neutralsodium salts, for example as the hexasodium salt of EDTMP or as thehepta- and octasodium salt of DTPMP. Here, preference is given to usingHEDP as builder from the class of phosphonates. In addition, theaminoalkane phosphonates have a marked heavy metal-binding capacity.Accordingly, particularly if the agents also contain bleaches, it may bepreferable to use aminoalkane phosphonates, in particular DTPMP, ormixtures of the said phosphonates.

In addition, all compounds which are able to form complexes withalkaline earth metal ions can be used as cobuilders.

The agents according to the invention may contain builder substances,where appropriate, in amounts of up to 90% by weight, and preferablycontain them in amounts of up to 75% by weight. Washing agents accordingto the invention have builder contents of, in particular, from 5% byweight to 50% by weight. In inventive agents for cleaning hard surfaces,in particular for machine cleaning of dishes, the builder substancecontent is in particular from 5% by weight to 88% by weight, withpreferably no water-insoluble builder materials being used in suchagents. A preferred embodiment of inventive agents for, in particular,machine cleaning of dishes contains from 20% by weight to 40% by weightwater-soluble organic builders, in particular alkali metal citrate, from5% by weight to 15% by weight alkali metal carbonate and from 20% byweight to 40% by weight alkali metal disilicate.

Solvents which may be used in the liquid to gelatinous compositions ofwashing and cleaning agents are, for example, from the group ofmonohydric or polyhydric alcohols, alkanolamines or glycol ethers, aslong as they are miscible with water in the given concentration range.Preferably, the solvents are selected from ethanol, n- or i-propanol,butanols, ethylene glycol methyl ether, ethylene glycol ethyl ether,ethylene glycol propyl ether, ethylene glycol mono-n-butyl ether,diethylene glycol methyl ether, diethylene glycol ethyl ether, propyleneglycol methyl, ethyl or propyl ether, dipropylene glycol monomethyl ormonoethyl ether, diisopropylene glycol monomethyl or monoethyl ether,methoxy, ethoxy or butoxy triglycol, 1-butoxyethoxy-2-propanol,3-methyl-3-methoxybutanol, propylene glycol t-butyl ether, and mixturesof these solvents.

Solvents may be used in the liquid to gelatinous washing and cleaningagents according to the invention in amounts of between 0.1 and 20% byweight, but preferably below 15% by weight, and in particular below 10%by weight.

To adjust the viscosity, one or more thickeners or thickening systemsmay be added to compositions according to the invention. These highmolecular weight substances which are also called swell(ing) agentsusually soak up the liquids and swell in the process, convertingultimately into viscous true or colloidal solutions.

Suitable thickeners are inorganic or polymeric organic compounds.Inorganic thickeners include, for example, polysilicic acids, clayminerals, such as montmorillonites, zeolites, silicas and bentonites.The organic thickeners are from the groups of natural polymers, modifiednatural polymers and completely synthetic polymers. Such naturalpolymers are, for example, agar-agar, carrageen, tragacanth, gum arabic,alginates, pectins, polyoses, guar flour, carob seed flour, starch,dextrins, gelatins and casein. Modified natural substances which areused as thickeners are primarily from the group of modified starches andcelluloses. Examples which may be mentioned here arecarboxymethylcellulose and other cellulose ethers, hydroxyethylcelluloseand hydroxypropylcellulose, and carob flour ether. Completely syntheticthickeners are polymers such as polyacrylic and polymethacryliccompounds, vinyl polymers, polycarboxylic acids, polyethers, polyimines,polyamides and polyurethanes.

The thickeners may be present in an amount up to 5% by weight,preferably from 0.05 to 2% by weight, and particularly preferably from0.1 to 1.5% by weight, based on the finished composition.

The washing and cleaning agent according to the invention may, whereappropriate, comprise, as further customary ingredients, sequesteringagents, electrolytes and further excipients.

The textile washing agents according to the invention may contain, asoptical brighteners, derivatives of diaminostilbenedisulfonic acid oralkali metal salts thereof. Suitable are, for example, salts of4,4′-bis(2-anilino-4-morpholino-1,3,5-triazinyl-6-amino)stilbene-2,2′-disulfonicacid or similarly constructed compounds which carry a diethanolaminogroup, a methylamino group, an anilino group or a 2-methoxyethylaminogroup instead of the morpholino group. In addition, brighteners of thesubstituted diphenylstyryl type may be present, for example the alkalimetal salts of 4,4′-bis(2-sulfostyryl)diphenyl,4,4′-bis(4-chloro-3-sulfostyryl)diphenyl, or4-(4-chlorostyryl)-4′-(2-sulfostyryl)diphenyl. Mixtures of theabovementioned optical brighteners may also be used.

Greying inhibitors have the function of keeping the soil detached fromthe textile fibre in suspension in the liquor. Suitable for this purposeare water-soluble colloids, usually organic in nature, for examplestarch, glue, gelatin, salts of ethercarboxylic acids or ethersulfonicacids of starch or of cellulose, or salts of acidic sulfuric esters ofcellulose or of starch. Water-soluble polyamides containing acidicgroups are also suitable for this purpose. Furthermore, starchderivatives other than those mentioned above may be used, for examplealdehyde starches. Preference is given to using cellulose ethers such ascarboxymethylcellulose (Na salt), methylcellulose, hydroxyalkylcelluloseand mixed ethers such as methylhydroxyethylcellulose,methylhydroxypropylcellulose, methylcarboxymethylcellulose, and mixturesthereof, for example in amounts of from 0.1 to 5% by weight, based onthe agents.

In order to protect against silver corrosion, silver corrosioninhibitors may be used in dishwashing cleaning agents according to theinvention. Such inhibitors are known in the prior art, for examplebenzotriazoles, iron(III) chloride or CoSO₄. As disclosed, silvercorrosion inhibitors which are particularly suitable for being usedtogether with enzymes are manganese, titanium, zirconium, hafnium,vanadium, cobalt, or cerium salts and/or complexes in which thespecified metals are present in any of the oxidation stages II, III, IV,V or VI. Examples of such compounds are MnSO₄, V₂O₅, V₂O₄, VO₂, TiOSO₄,K₂TiF₆, K₂ZrF₆, Co(NO₃)₂, Co(NO₃)₃, and mixtures thereof.

Soil-release active ingredients or soil repellents are usually polymerswhich, when used in a washing agent, impart soil-repellent properties tothe laundry fibre and/or assist the ability of the other washing agentingredients to detach soil. A comparable effect can also be observedwith their use in cleaning agents for hard surfaces.

Soil-release active ingredients which are particularly effective andhave been known for a long time are copolyesters having dicarboxylicacid, alkylene glycol and polyalkylene glycol units. Examples thereofare copolymers or mixed polymers of polyethylene terephthalate andpolyoxyethylene glycol and acidic agents containing, inter alia, acopolymer of a dibasic carboxylic acid and an alkylene or cycloalkylenepolyglycol. Polymers of ethylene terephthalate and polyethylene oxideterephthalate and also copolyesters of ethylene glycol, polyethyleneglycol, aromatic dicarboxylic acid and sulfonated aromatic dicarboxylicacid in particular molar ratios may also be used advantageously. Alsoknown are methyl or ethyl group end-group-capped polyesters havingethylene and/or propylene terephthalate and polyethylene oxideterephthalate units, and washing agents containing such a soil-releasepolymer, and also polyesters which contain, in addition to oxyethylenegroups and terephthalic acid units, also substituted ethylene units andglycerol units. Also known are polyesters which contain, in addition tooxyethylene groups and terephthalic acid units, 1,2-propylene,1,2-butylene and/or 3-methoxy-1,2-propylene groups, and glycerol unitsand which are end-group-capped with C₁- to C₄-alkyl groups, and alsopolyesters having polypropylene terephthalate and polyoxyethyleneterephthalate units, which are at least partially end-group-capped byC₁₋₄-alkyl or acyl radicals. Also known are sulfoethyl end-group-cappedterephthalate-containing soil-release polyesters, and soil-releasepolyesters having terephthalate, alkylene glycol and poly-C₂₋₄-glycolunits, produced by sulfonation of unsaturated end groups. Othersubstances which have been disclosed are acidic, aromatic polyesterscapable of detaching soil, and nonpolymeric soil-repellent activeingredients for materials made of cotton, which have a plurality offunctional units: a first unit which may be cationic, for example, isable to adsorb to the cotton surface by means of electrostaticinteraction, and a second unit which is hydrophobic is responsible forthe active ingredient remaining at the water/cotton interface.

The color transfer inhibitors suitable for use in textile washing agentsaccording to the invention include, in particular,polyvinylpyrrolidones, polyvinylimidazoles, polymeric N-oxides such aspoly(vinylpyridine N-oxide) and copolymers of vinylpyrrolidone withvinylimidazole.

For use in machine cleaning processes, it may be of advantage to addfoam inhibitors to the agents. Examples of suitable foam inhibitors aresoaps of natural or synthetic origin having a high proportion of C₁₈-C₂₄fatty acids. Examples of suitable nonsurfactant-type foam inhibitors areorganopolysiloxanes and their mixtures with microfine, optionallysilanized silica and also paraffins, waxes, microcrystalline waxes, andmixtures thereof with silanized silica or bis-stearyl-ethylenediamide.With advantages, use is also made of mixtures of different foaminhibitors, for example mixtures of silicones, paraffins or waxes. Thefoam inhibitors, in particular those containing silicone and/orparaffin, are preferably bound to a granular, water-soluble ordispersible support substance. Particular preference is given here tomixtures of paraffins and bis-stearylethylenediamides.

The application DE 102004020430.6 which has not been publishedpreviously reveals cleansers, in particular machine dishwashing agents,which contain a copolymer of (i) unsaturated carboxylic acids, (ii)monomers containing sulfonic acid groups and (iii) optionally furtherionic or nonionogenic monomers and a special α-amylase variant. Saidcopolymers can also be combined with α-amylases according to theinvention, in particular if the latter, in addition to the substitutionsaccording to the invention, have substitutions of the kind that can befound in one or more of the applications WO 96/23873 A1, WO 00/60060 A2and WO 01/66712 A2. This applies in particular if the commercial productStainzyme® from Novozymes, which falls under these applications, isimproved in further positions and is additionally provided with at leastone substitution of the invention. This is because an additive effect ofthe various modifications must be assumed in principle.

A cleaning agent according to the invention for hard surfaces may, inaddition, contain ingredients with abrasive action, in particular fromthe group comprising quartz flours, wood flours, polymer flours, chalksand glass microbeads, and mixtures thereof. Abrasives are present in thecleaning agents according to the invention preferably at not more than20% by weight, in particular from 5% by weight to 15% by weight.

Dyes and fragrances are added to washing and cleaning agents in order toimprove the aesthetic appeal of the products and to provide theconsumer, in addition to washing and cleaning performance, with avisually and sensorially “typical and unmistakable” product. As perfumeoils and/or fragrances it is possible to use individual odorantcompounds, for example the synthetic products of the ester, ether,aldehyde, ketone, alcohol and hydrocarbon types. Odorant compounds ofthe ester type are, for example, benzyl acetate, phenoxyethylisobutyrate, p-tert-butylcyclohexyl acetate, linalyl acetate,dimethylbenzylcarbinyl acetate, phenylethyl acetate, linalyl benzoate,benzyl formate, ethyl methylphenyl glycinate, allylcyclohexylpropionate, styrallyl propionate and benzyl salicylate. The ethersinclude, for example, benzyl ethyl ether; the aldehydes include, forexample, the linear alkanals having 8-18 carbon atoms, citral,citronellal, citronellyloxyacetaldehyde, cyclamenaldehyde,hydroxycitronellal, lilial and bourgeonal; the ketones include, forexample, the ionones, α-isomethylionone and methyl cedryl ketone; thealcohols include anethol, citronellol, eugenol, geraniol, linalool,phenylethyl alcohol, and terpineol; the hydrocarbons include primarilythe terpenes such as limonene and pinene. Preference, however, is givento the use of mixtures of different odorants which together produce anappealing fragrance note. Such perfume oils may also contain naturalodorant mixtures, as obtainable from plant sources, for example pineoil, citrus oil, jasmine oil, patchouli oil, rose oil or ylang-ylangoil. Likewise suitable are muscatel, sage oil, camomile oil, clove oil,balm oil, mint oil, cinnamon leaf oil, lime blossom oil, juniper berryoil, vetiver oil, olibanum oil, galbanum oil and labdanum oil, and alsoorange blossom oil, neroli oil, orangepeel oil and sandalwood oil. Thedye content of washing and cleaning agents is usually less than 0.01% byweight, while fragrances may make up up to 2% by weight of the overallformulation.

The fragrances may be incorporated directly into the washing andcleaning agents; however, it may also be advantageous to apply thefragrances to carriers which intensify the adhesion of the perfume tothe material to be cleaned and, by means of slower fragrance release,ensure long-lasting fragrance, in particular of treated textiles.Materials which have become established as such carriers are, forexample, cyclodextrins, it being possible, in addition, for thecyclodextrin-perfume complexes to be additionally coated with furtherauxiliaries. Another preferred carrier for fragances is the describedzeolite X which can also absorb fragrances instead of or in a mixturewith surfactants. Preference is therefore given to washing and cleaningagents which contain the described zeolite X and fragrances which,preferably, are at least partially absorbed on the zeolite.

Preferred dyes whose selection is by no means difficult for the skilledworker have high storage stability and insensitivity to the otheringredients of the agents and to light, and also have no pronouncedaffinity for textile fibres, so as not to stain them.

To control microorganisms, washing or cleaning agents may containantimicrobial active ingredients. Depending on antimicrobial spectrumand mechanism of action, a distinction is made here betweenbacteriostatics and bactericides, fungistatics and fungicides, etc.Examples of important substances from these groups are benzalkoniumchlorides, alkylaryl sulfonates, halogen phenols and phenol mercuryacetate. The terms antimicrobial action and antimicrobial activeingredient have, within the teaching according to the invention, themeaning common in the art. Suitable antimicrobial active ingredients arepreferably selected from the groups of alcohols, amines, aldehydes,antimicrobial acids or their salts, carboxylic esters, acid amides,phenols, phenol derivatives, diphenyls, diphenylalkanes, ureaderivatives, oxygen acetals, nitrogen acetals and also oxygen andnitrogen formals, benzamidines, isothiazolines, phthalimide derivatives,pyridine derivatives, antimicrobial surfactant compounds, guanidines,antimicrobial amphoteric compounds, quinolines,1,2-dibromo-2,4-dicyanobutane, iodo-2-propylbutyl carbamate, iodine,iodophors, peroxo compounds, halogen compounds, and any mixtures of theabove.

The antimicrobial active ingredient may be selected from ethanol,n-propanol, isopropanol, 1,3-butanediol, phenoxyethanol, 1,2-propyleneglycol, glycerol, undecylenic acid, benzoic acid, salicylic acid,dihydracetic acid, o-phenylphenol, N-methylmorpholinoacetonitrile (MMA),2-benzyl-4-chlorophenol, 2,2′-methylenebis(6-bromo-4-chlorophenol),4,4′-dichloro-2′-hydroxydiphenyl ether (dichlosan),2,4,4′-trichloro-2′-hydroxydiphenyl ether (trichlosan), chlorohexidine,N-(4-chlorophenyl)-N-(3,4-dichlorophenyl)urea, N,N′-(1,10-decanediyldi-1-pyridinyl-4-ylidene)-bis(1-octanamine)dihydrochloride, N,N′-bis(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetra-azatetradecanediimidamide,glucoprotamines, antimicrobial surface-active quaternary compounds,guanidines including the bi- and polyguanidines, such as, for example,1,6-bis(2-ethylhexylbiguanidohexane) dihydrochloride,1,6-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)hexane tetrahydrochloride,1,6-di-(N₁,N₁′-phenyl-N₁,N₁-methyldiguanido-N₅,N₅′)hexanedihydrochloride, 1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅,N₅′)hexanedihydrochloride,1,6-di-(N₁,N₁′-2,6-dichlorophenyldiguanido-N₅,N₅′)hexanedihydrochloride,1,6-di-[N₁,N₁′-beta-(p-methoxyphenyl)diguanido-N₅,N₅′]hexanedihydrochloride,1,6-di-(N₁,N₁′-alpha-methyl-beta-phenyldiguanido-N₅,N₅′)hexanedihydrochloride, 1,6-di-(N₁,N₁′-p-nitrophenyldiguanido-N₅,N₅′)hexanedihydrochloride, omega:omega-di-(N₁,N₁phenyldiguanido-N₅,N₅′)-di-n-propyl ether dihydrochloride,omega:omega′-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)-di-n-propylether tetrahydrochloride,1,6-di-(N₁,N₁′-2,4-dichlorophenyldiguanido-N₅,N₅′)hexanetetrahydrochloride, 1,6-di-(N₁,N₁′-p-methylphenyldiguanido-N₅,N₅′)hexanedihydrochloride,1,6-di-(N₁,N₁′-2,4,5-trichlorophenyldiguanido-N₅,N₅′)hexanetetrahydrochloride,1,6-di-[N₁,N₁′-alpha-(p-chlorophenyl)ethyldiguanido-N₅,N₅′]hexanedihydrochloride,omega:omega-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)_(m)-xylenedihydrochloride, 1,12-di-(N₁,N₁′-p-chlorophenyldiguanido-N₅,N₅′)dodecanedihydrochloride, 1,10-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)decanetetrahydrochloride, 1,12-di-(N₁,N₁′-phenyldiguanido-N₅,N₅′)dodecanetetrahydrochloride, 1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅,N₅′)hexanedihydrochloride, 1,6-di-(N₁,N₁′-o-chlorophenyldiguanido-N₅, N₅′)hexanetetrahydrochloride, ethylene-bis(1-tolylbiguanide),ethylene-bis(p-tolylbiguanide),ethylene-bis(3,5-dimethylphenylbiguanide),ethylene-bis(p-tert-amylphenylbiguanide),ethylene-bis(nonylphenylbiguanide), ethylene-bis(phenylbiguanide),ethylene-bis(N-butylphenylbiguanide),ethylene-bis(2,5-diethoxyphenylbiguanide),ethylene-bis(2,4-dimethylphenylbiguanide),ethylene-bis(o-diphenylbiguanide), ethylene-bis(mixed amylnaphthylbiguanide), N-butylethylene-bis(phenylbiguanide),trimethylenebis(o-tolylbiguanide),N-butyl-trimethyl-bis(phenylbiguanide) and the corresponding salts suchas acetates, gluconates, hydrochlorides, hydrobromides, citrates,bisulfites, fluorides, polymaleates, N-cocoalkyl sarcosinates,phosphites, hypophosphites, perfluorooctanoates, silicates, sorbates,salicylates, maleates, tartrates, fumarates,ethylenediaminetetraacetates, iminodiacetates, cinnamates, thiocyanates,arginates, pyromellitates, tetracarboxybutyrates, benzoates, glutarates,monofluorophosphates, perfluoropropionates, and any mixtures thereof.Also suitable are halogenated xylene and cresol derivatives, such asp-chlorometacresol or p-chlorometaxylene, and natural antimicrobialactive ingredients of plant origin (for example from spices or herbs),animal origin and microbial origin. Preference may be given to usingantimicrobial surface-active quaternary compounds, a naturalantimicrobial active ingredient of plant origin and/or a naturalantimicrobial active ingredient of animal origin, most preferably atleast one natural antimicrobial active ingredient of plant origin fromthe group comprising caffeine, theobromine and theophylline andessential oils such as eugenol, thymol and geraniol, and/or at least onenatural antimicrobial active ingredient of animal origin from the groupcomprising enzymes such as milk protein, lysozyme and lactoperoxidase,and/or at least one antimicrobial surface-active quaternary compoundhaving an ammonium, sulfonium, phosphonium, iodonium or arsonium group,peroxo compounds and chlorine compounds. It is also possible to usesubstances of microbial origin, the “bacteriocines”.

The quaternary ammonium compounds (QACS) which are suitable asantimicrobial active ingredients have the general formula(R¹)(R²)(R³)(R⁴)N⁺X⁻ where R¹ to R⁴ are identical or differentC₁-C₂₂-alkyl radicals, C₇-C₂₈-aralkyl radicals or heterocyclic radicals,where two, or in the case of an aromatic incorporation as in pyridine,even three radicals, together with the nitrogen atom, form theheterocycle, for example a pyridinium or imidazolinium compound, and X⁻are halide ions, sulfate ions, hydroxide ions or similar anions. Foroptimal antimicrobial action, at least one of the radicals preferablyhas a chain length of from 8 to 18, in particular 12 to 16, carbonatoms.

QACs can be prepared by reacting tertiary amines with alkylating agentssuch as, for example, methyl chloride, benzyl chloride, dimethylsulfate, dodecyl bromide, or else ethylene oxide. The alkylation oftertiary amines having one long alkyl radical and two methyl groupsproceeds particularly readily, and the quaternization of tertiary amineshaving two long radicals and one methyl group can also be carried outwith the aid of methyl chloride under mild conditions. Amines which havethree long alkyl radicals or hydroxy-substituted alkyl radicals have lowreactivity and are preferably quaternized using dimethyl sulfate.

Examples of suitable QACs are benzalkonium chloride (N-alkyl-N,N-dimethylbenzylammonium chloride, CAS No. 8001-54-5), benzalkone B(m,p-dichlorobenzyldimethyl-C12-alkylammonium chloride, CAS No.58390-78-6), benzoxonium chloride(benzyldodecyl-bis(2-hydroxyethyl)ammonium chloride), cetrimoniumbromide (N-hexadecyl-N, N-trimethylammonium bromide, CAS No. 57-09-0),benzetonium chloride (N,N-dimethyl-N-[2-[2-[p-(1,1,3,3-tetramethylbutyl)phenoxy]ethoxy]ethyl]benzylammoniumchloride, CAS No. 121-54-0), dialkyldimethylammonium chlorides such asdi-n-decyldimethylammonium chloride (CAS No. 7173-51-5-5),didecyldimethylammonium bromide (CAS No. 2390-68-3),dioctyldimethylammonium chloride, 1-cetylpyridinium chloride (CAS No.123-03-5) and thiazoline iodide (CAS No. 15764-48-1), and mixturesthereof. Particularly preferred QACs are the benzalkonium chlorideshaving C₈-C₁₈-alkyl radicals, in particularC₁₂-C₁₄-alkylbenzyldimethylammonium chloride.

Benzalkonium halides and/or substituted benzalkonium halides arecommercially available, for example, as Barquat® ex Lonza, Marquat® exMason, Variquat® ex Witco/Sherex and Hyamine® ex Lonza, and Bardac® exLonza. Further commercially available antimicrobial active ingredientsare N-(3-chloroallyl)hexaminium chloride such as Dowicide® and Dowicil®ex Dow, benzethonium chloride such as Hyamine® 1622 ex Rohm & Haas,methylbenzethonium chloride such as Hyamine® 10X ex Rohm & Haas,cetylpyridinium chloride such as cepacol chloride ex Merrell Labs.

The antimicrobial active ingredients are used in amounts of from 0.0001%by weight to 1% by weight, preferably from 0.001% by weight to 0.8% byweight, particularly preferably from 0.005% by weight to 0.3% by weight,and in particular from 0.01 to 0.2% by weight.

The agents may contain UV absorbers which attach to the treated textilesand improve the light stability of the fibres and/or the light stabilityof other formulation constituents. UV absorbers mean organic substances(light protection filters) which are able to absorb ultravioletradiation and to emit the absorbed energy again in the form of radiationof longer wavelength, for example heat.

Compounds which have these desired properties are, for example, thecompounds which are active via radiationless deactivation andderivatives of benzophenone having substitutents in position(s) 2 and/or4. Furthermore, also suitable are substituted benzotriazoles, acrylateswhich are phenyl-substituted in position 3 (cinnamic acid derivatives,with or without cyano groups in position 2), salicylates, organic Nicomplexes and natural substances such as umbelliferone and theendogenous urocanic acid. Of particular importance are biphenyl andespecially stilbene derivatives, for example those commerciallyavailable as Tinosorb® FD or Tinosorb® FR ex Ciba. UV-B absorbers whichmay be mentioned are: 3-benzylidenecamphor or 3-benzylidenenorcamphorand derivatives thereof, for example 3-(4-methylbenzylidene)camphor;4-aminobenzoic acid derivatives, preferably 2-ethylhexyl4-(dimethylamino)benzoate, 2-octyl 4-(dimethylamino)benzoate and amyl4-(dimethylamino)benzoate; esters of cinnamic acid, preferably2-ethylhexyl 4-methoxycinnamate, propyl 4-methoxycinnamate, isoamyl4-methoxycinnamate, 2-ethylhexyl 2-cyano-3,3-phenylcinnamate(octocrylenes); esters of salicylic acid, preferably 2-ethylhexylsalicylate, 4-isopropylbenzyl salicylate, homomenthyl salicylate;derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone,2-hydroxy-4-methoxy-4′-methylbenzophenone,2,2′-dihydroxy-4-methoxybenzophenone; esters of benzalmalonic acid,preferably di-2-ethylhexyl 4-methoxybenzmalonate; triazine derivativessuch as, for example,2,4,6-trianilino-(p-carbo-2′-ethyl-1′-hexyloxy)-1,3,5-triazine andoctyltriazone, or dioctylbutamidotriazones (Uvasorb® HEB);propane-1,3-diones such as, for example,1-(4-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione;ketotricyclo(5.2.1.0)decane derivatives. Further suitable are2-phenylbenzimidazole-5-sulfonic acid and its alkali metal, alkalineearth metal, ammonium, alkylammonium, alkanolammonium and glucammoniumsalts; sulfonic acid derivatives of benzophenones, preferably2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and its salts; sulfonicacid derivatives of 3-benzylidenecamphor, such as, for example,4-(2-oxo-3-bornylidenemethyl)benzenesulfonic acid and2-methyl-5-(2-oxo-3-bornylidene)sulfonic acid and salts thereof.

Suitable typical UV-A filters are, in particular, derivatives ofbenzoylmethane, such as, for example,1-(4′-tert-butylphenyl)-3-(4′-methoxyphenyl)propane-1,3-dione,4-tert-butyl-4′-methoxydibenzoylmethane (Parsol 1789),1-phenyl-3-(4′-isopropylphenyl)propane-1,3-dione, and enamine compounds(BASF). The UV-A and UV-B filters may of course also be used inmixtures. In addition to the said soluble substances, insoluble lightprotection pigments, namely finely dispersed, preferably nanoized, metaloxides or salts, are also suitable for this purpose. Examples ofsuitable metal oxides are, in particular, zinc oxide and titaniumdioxide and also oxides of iron, zirconium, silicon, manganese,aluminium and cerium, and mixtures thereof. Salts which may be used aresilicates (talc), barium sulfate or zinc stearate. The oxides and saltsare already used in the form of the pigments for skin-care andskin-protective emulsions and decorative cosmetics. The particles hereshould have an average diameter of less than 100 nm, preferably between5 and 50 nm, and in particular between 15 and 30 nm. They can have aspherical shape, but it is also possible to use particles which have anellipsoidal shape or a shape deviating in some other way from thespherical form. The pigments may also be surface-treated, i.e.hydrophilicized or hydrophobicized. Typical examples are coated titaniumdioxides such as, for example, titanium dioxide T 805 (Degussa) orEusolex® T2000 (Merck); suitable hydrophobic coating agents are herepreferably silicones and, particularly preferably, trialkoxyoctylsilanesor simethicones. Preference is given to using micronized zinc oxide.

The UV absorbers are usually used in amounts of from 0.01% by weight to5% by weight, preferably from 0.03% by weight to 1% by weight.

To increase the washing or cleaning performance, agents according to theinvention may contain further enzymes in addition to the α-amylasevariants of the invention, wherein it is possible in principle to useany enzymes established for these purposes in the prior art. Theseinclude in particular proteases, further amylases, lipases,hemicellulases, cellulases or oxidoreductases such as peroxidases and/orperhydrolases, and preferably mixtures thereof. These enzymes are inprinciple of natural origin; starting from the natural molecules,improved variants for use in detergents and cleansers are availablewhich are used with preference accordingly. Agents according to theinvention preferably contain enzymes in total amounts of from 1×10⁻⁸ to5 percent by weight, based on active protein. The protein concentrationmay be determined with the aid of known methods, for example the BCAmethod (bicinchonic acid; 2,2′-biquinolyl-4,4′-dicarboxylic acid) or thebiuret method (A. G. Gornall, C. S. Bardawill and M. M. David, J. Biol.Chem., 177 (1948), pp. 751-766).

Among the proteases, preference is given to those of the subtilisintype. Examples thereof include the subtilisins BPN′ and Carlsberg,protease PB92, subtilisins 147 and 309, Bacillus lentus alkalineprotease, subtilisin DY and the enzymes thermitase, proteinase K and theproteases TW3 and TW7, all of which can be classified to the subtilasesbut no longer to the subtilisins in the narrower sense. SubtilisinCarlsberg is available in a developed form under the trade nameAlcalase® from Novozymes A/S, Bagsvaerd, Denmark. Subtilisins 147 and309 are sold under the trade names Esperase®, and Savinase®,respectively, from Novozymes. The variants listed under the name BLAP®are derived from the protease of Bacillus lentus DSM 5483 (WO 91/02792A1) and are described in particular in WO 92/21760 A1, WO 95/23221 A1,WO 02/088340 A2 and WO 03/038082 A2. The applications WO 03/054185 A1,WO 03/056017 A2, WO 03/055974 A2 and WO 03/054184 A1 reveal furtherusable proteases from various Bacillus sp. and B. gibsonii.

Further examples of useful proteases are the enzymes available under thetrade names Durazym®, Relase®, Everlase®, Nafizym®, Natalase®, Kannase®and Ovozymes® from Novozymes, those under the trade names Purafect®,Purafect® OxP and Properase® from Genencor, that under the trade nameProtosol® from Advanced Biochemicals Ltd, Thane, India, that under thetrade name Wuxi® from Wuxi Snyder Bioproducts Ltd., China, those underthe trade names Proleather® and Protease P® from Amano PharmaceuticalsLtd., Nagoya, Japan, and known as proteinase K-16 from Kao Corp., Tokyo,Japan.

Examples of amylases which may be employed additionally according to theinvention are the α-amylases of Bacillus licheniformis, of B.amyloliquefaciens or of B. stearothermophilus, and developments thereofwhich have been improved for use in detergents and cleansers. The B.licheniformis enzyme is available from Novozymes under the nameTermamyl® and from Genencor under the name Purastar® ST. Developmentproducts of this α-amylase are available from Novozymes, under the namesDuramyl® and Termamyl® ultra, from Genencor, under the name Purastar®OxAm, and from Daiwa Seiko Ink., Tokyo Japan, as Keistase®. The B.amyloliquefaciens α-amylase is sold by Novozymes under the name BAN®,and variants derived from the B. stearothermophilus α-amylase are soldunder the names BSG® and Novamyl®, likewise from Novozymes.

Enzymes which should additionally be emphasized for this purpose are theα-amylase from Bacillus sp. A 7-7 (DSM 12368) disclosed in theapplication WO 02/10356 A2 and the cyclodextrin glucanotransferase(CGTase) from B. agaradherens (DSM 9948), described in the applicationWO 02/44350. It is further possible to use the amylolytic enzymes whichbelong to the sequence space of α-amylases, which is defined in theapplication WO 03/002711 A2, and those described in the application WO03/054177 A2. It is also possible to use fusion products of themolecules mentioned, for example those of the application DE 10138753A1, or point mutations thereof.

Also suitable are the developments of α-amylase from Aspergillus nigerand A. oryzae, which are available under the trade name Fungamyl® fromNovozymes. Other examples of commercial products which may be used areAmylase-LT® and Stainzyme®, the latter of which likewise from Novozymes.

Agents of the invention may comprise lipases or cutinases, in particulardue to their triglyceride-cleaving activities, but also in order togenerate peracids in situ from suitable precursors. Examples thereofinclude the lipases which were originally derived from Humicolalanuginosa (Thermomyces lanufinosus) or have been developed, inparticular those having the D96L amino acid substitution. They are sold,for example, under the trade names Lipolase®, Lipolase® Ultra,LipoPrime®, Lipozyme® and Lipex® by Novozymes. It is furthermorepossible to use, for example, the cutinases which have originally beenisolated from Fusarium solani pisi and Humicola insolens. Lipases whichare also useful can be obtained under the names Lipase CE®, Lipase P®,Lipase B®, or Lipase CES®, Lipase AKG®, Bacillus sp. Lipase®, LipasaeAP®, Lipase M-AP® and Lipase AML® from Amano. Examples of lipases andcutinases from Genencor, which can be used, are those whose startingenzymes have originally been isolated from Pseudomonas mendocina andFusarium solanii. Other important commercial products which may bementioned are the Ml Lipase® and Lipomax® preparations originally soldby Gist-Brocades and the enzymes sold under the names Lipase MY-30®,Lipase OF® and Lipase PL® by Meito Sangyo KK, Japan, and also theproduct Lumafast® from Genencor.

Agents according to the invention may, in particular when intended forthe treatment of textiles, comprise cellulases, depending on the purposeeither as pure enzymes, as enzyme preparations or in the form ofmixtures in which the individual components advantageously complementone another with respect to their different performance aspects. Theseperformance aspects include in particular contributions to the primarywashing performance, to the secondary washing performance of the agent(antiredeposition action or graying inhibition) and finishing (fabricaction), up to exerting a “stonewashed” effect.

A useful fungal, endoglucanase (EG)-rich cellulase preparation anddevelopments thereof are supplied under the tradename Celluzyme® byNovozymes. The products Endolase® and Carezyme®, likewise available fromNovozymes, are based on the H. insolens DSM 1800 50 kD EG and 43 kD EGrespectively. Further commercial products of this company, which may beused, are Cellusoft® and Renozyme®. The latter is based on theapplication WO 96/29397 A1. Performance enhanced cellulase variants canbe found, for example, in the application WO 98/12307 A1. It is alsopossible to use the cellulases disclosed in the application WO 97/14804A1; for example the Melanocarpus 20 kD EG disclosed therein, which isavailable from AB Enzymes, Finland, under the trade names Ecostone® andBiotouch®. Further commercial products from AB Enzymes are Econase® andEcopulp®. WO 96/34092 A2 discloses further suitable cellulases fromBacillus sp. CBS 670.93 and CBS 669.93, with that of Bacillus sp. CBS670.93 being available under the trade name Puradax® from Genencor.Other commercial products from Genencor are “Genencor detergentcellulase L” and IndiAge® Neutra.

Agents according to the invention may comprise further enzymes, inparticular for removing particular problematic soilings, which arecombined under the term hemicellulases. These include, for example,mannanases, xanthane lyases, pectin lyases (=pectinases), pectinesterases, pectate lyases, xyloglucanases (=xylanases), pullulanases andβ-glucanases. Examples of suitable mannanases are available under thenames Gamanase® and Pektinex AR® from Novozymes, under the name Rohapec®B1L from AB Enzymes, under the name Pyrolase® from Diversa Corp., SanDiego, Calif., USA, and under the name Purabrite® from Genencor Int.,Inc., Palo Alto, Calif., USA. An example of a suitable β-glucanase froma B. alcalophilus is revealed by the application WO 99/06573 A1. Theβ-glucanase obtained from B. subtilis is available under the nameCereflo® from Novozymes.

To enhance the bleaching action, detergents and cleansers according tothe invention may comprise oxidoreductases, for example oxidases,oxygenases, katalases, peroxidases such as haloperoxidases,chloroperoxidases, bromoperoxidases, lignin peroxidases, glucoseperoxidases or manganese peroxidases, dioxygenases or laccases (phenoloxidases, polyphenol oxidases). Suitable commercial products which maybe mentioned are Denilite® 1 and 2 from Novozymes. Reference may be madeto the application WO 98/45398 A1 with respect to examples of systemsfor an enzymic perhydrolysis, which may be used advantageously. WO2004/058955 A2, for example, discloses choline oxidases useful inparticular for such a system. The application WO 2004/058961 A1 revealsmodified proteases having a pronounced perhydrolase activity which maylikewise be used advantageously here, in particular for achieving mildbleaching in detergents for textiles. The application DE 102004029475.5describes a combined enzymic bleaching system comprising an oxidase anda perhydrolase. WO 2005/056782 A2 also discloses further perhydrolaseswhich may be used according to the invention. Advantageously, preferablyorganic, more preferably aromatic, compounds which interact with theenzymes are additionally added in order to enhance the activity of theoxidoreductases in question (enhancers), or to ensure the electron fluxin the event of large differences in the redox potentials of theoxidizing enzymes and the soilings (mediators).

The enzymes used in agents according to the invention either originatefrom microorganisms, for example of the genera Bacillus, Streptomyces,Humicola, or Pseudomonas, and/or are produced in biotechnology processesknown per se by suitable microorganisms, for example by transgenicexpression hosts of the Bacillus genera or by filamentous fungi.

The enzymes in question are favorably purified via processes which areestablished per se, for example via precipitation, sedimentation,concentration, filtration of the liquid phases, microfiltration,ultrafiltration, the action of chemicals, deodorization or suitablecombinations of these steps.

The enzymes may be added to the agents according to the invention in anyform established in the prior art, including, for example, the solidpreparations obtained by granulation, extrusion or lyophilization, or,in particular in the case of liquid or gel-like agents, solutions of theenzymes, advantageously highly concentrated, low in water and/or admixedwith stabilizers.

Alternatively, the enzymes may be encapsulated both for solid and liquidforms of presentation, for example by spray-drying or extrusion of theenzyme solution together with a, preferably natural, polymer, or in theform of capsules, for example those in which the enzymes are enclosed asin a solidified gel, or in those of the core-shell type, in which anenzyme-containing core is coated with a water-, air- and/orchemical-impermeable protective layer. It is possible to additionallyapply further active ingredients, for example stabilizers, emulsifiers,pigments, bleaches or dyes, in the form of layers applied thereupon.Such capsules are applied by methods known per se, for example byagitated or roll granulation or in fluidized bed processes.Advantageously, such granules are low-dusting, for example due toapplication of polymeric film formers, and storage-stable as a result ofsaid coating.

It is furthermore possible to formulate two or more enzymes together, sothat a single granule has a plurality of enzyme activities.

A protein and/or enzyme present in an agent of the invention may beprotected, particularly during storage, from damage such as, forexample, inactivation, denaturation or decay, for instance due tophysical influences, oxidation or proteolytic cleavage. When saidproteins and/or enzymes are produced microbially, particular preferenceis given to inhibiting proteolysis, in particular when the agents alsocomprise proteases. For this purpose, preferred agents according to theinvention comprise stabilizers.

One group of stabilizers are reversible protease inhibitors. Frequently,benzamidine hydrochloride, borax, boric acids, boronic acids or salts oresters thereof are used, and of these especially derivatives havingaromatic groups, for example ortho-, meta- or para-substitutedphenylboronic acids, in particular 4-formylphenylboronic acid, or thesalts or esters of said compounds. Peptide aldehydes, i.e. oligopeptideswith reduced C terminus, in particular those composed of from 2 to 50monomers, are also used for this purpose. Peptidic reversible proteaseinhibitors include inter alia ovomucoid and leupeptin. Specific,reversible peptide inhibitors of the protease subtilisin and also fusionproteins of proteases and specific peptide inhibitors are also suitablefor this.

Further enzyme stabilizers are amino alcohols such as mono-, di-,triethanol- and -propanolamine and mixtures thereof, aliphaticcarboxylic acids up to C₁₂, such as succinic acid for example, otherdicarboxylic acids or salts of said acids. End group-capped fatty acidamide alkoxylates are also suitable for this purpose. Particular organicacids used as builders are capable of additionally stabilizing an enzymepresent, as disclosed in WO 97/18287.

Lower aliphatic alcohols, but especially polyols such as, for example,glycerol, ethylene glycol, propylene glycol or sorbitol, are otherfrequently used enzyme stabilizers. Diglycerol phosphate also protectsagainst denaturation due to physical influences. Calcium salts and/ormagnesium salts are also used, for example calcium acetate or calciumformate.

Polyamide oligomers or polymeric compounds such as lignin, water-solublevinyl copolymers or cellulose ethers, acrylic polymers and/orpolyamides, stabilize the enzyme preparation inter alia to physicalinfluences or pH fluctuations. Polyamine N-oxide-containing polymers actsimultaneously as enzyme stabilizers and as color transfer inhibitors.Other polymeric stabilizers are the linear C₈-C₁₈ polyoxyalkylenes.Alkylpolyglycosides can likewise stabilize the enzymic components of theagent of the invention and are preferably able to increase in additionthe performance of said components. Crosslinked N-containing compoundsfulfill a double function as soil release agents and as enzymestabilizers. Hydrophobic, nonionic polymer stabilizes in particular anoptionally present cellulase.

Reducing agents and antioxidants increase the stability of the enzymesto oxidative decay; familiar examples thereof are sulfur-containingreducing agents. Other examples are sodium sulfite and reducing sugars.

Particular preference is given to using combinations of stabilizers, forexample of polyols, boric acid and/or borax, the combination of boricacid or borate, reducing salts and succinic acid or other dicarboxylicacids, or the combination of boric acid or borate with polyols orpolyamino compounds and with reducing salts. The action ofpeptide-aldehyde stabilizers can be increased advantageously bycombination with boric acid and/or boric acid derivatives and polyols,and furthermore by the additional action of divalent cations, forexample calcium ions.

In a preferred embodiment, agents according to the invention arecharacterized in that they are composed of more than one phase, forexample in order to release the active ingredients separately from oneanother with regard to time or space. Said phases may be in variousstates of aggregation but in particular in the same state ofaggregation.

Agents according to the invention which are composed of more than onesolid component may be prepared in a simple manner by mixing varioussolid components, in particular powder, granules or extrudates, havingvarious ingredients and/or different release behavior with one anotherin an overall loose mixture. Solid agents according to the inventionwhich consist of one or more phases may be prepared in the known manner,for example by spray-drying or granulation, adding the enzymes andpossible further thermosensitive ingredients such as, for example,bleaches, separately later, where appropriate. To prepare agentsaccording to the invention having an increased bulk density, inparticular in the range from 650 g/l to 950 g/l, preference is given toa method which has an extrusion step and has been disclosed in Europeanpatent EP 0 486 592. European patent EP 0 642 576 describes anotherpreferred preparation with the aid of a granulation process.

Proteins may be used in dried, granulated, encapsulated, or encapsulatedand additionally dried form, for example, for solid agents. They may beadded separately, i.e. as a separate phase, or together with othercomponents in the same phase, with or without compaction. Ifmicroencapsulated enzymes are to be processed in solid form, it ispossible to remove the water from the aqueous solutions resulting fromthe workup by using methods known in the prior art, such asspray-drying, removing by centrifugation or resolubilizing. Theparticles obtained in this way are usually between 50 and 200 μm insize.

The encapsulated form is a way of protecting the enzymes from othercomponents such as, for example, bleaches, or of making a controlledrelease possible. Depending on their size, the capsules are divided intomilli-, micro- and nanocapsules, microcapsules being particularlypreferred for enzymes. Another possible encapsulation method is toencapsulate the enzymes suitable for use in detergents or cleansers,starting from a mixture of the enzyme solution with a solution orsuspension of starch or a starch derivative, into starch or said starchderivative.

It is also possible for least two phases to be associated with oneanother. Thus, compression or compacting to give tablets is anotherpossibility of providing a solid agent according to the invention. Suchtablets may have one or more phases. Consequently, this presentationform also offers the possibility of providing a two-phase solid agentaccording to the invention. To produce agents according to the inventionin tablet form, which may have one or more phases, may have one or morecolors and/or consist of one or more layers, preference is given tomixing all of the components—per one layer, where appropriate—with oneanother in a mixer and compressing said mixture by means of conventionaltabletting presses, for example eccentric presses or rotary presses, atpressing forces in the range of from about 50 to 100 kN/cm², preferablyat from 60 to 70 kN/cm². Particularly in the case of multilayer tablets,it may be of advantage if at least one layer is compressed beforehand.This is preferably accomplished at pressing forces of between 5 and 20kN/cm², in particular at from 10 to 15 kN/cm². A tablet produced in thisway preferably has a weight of from 10 g to 50 g, in particular from 15g to 40 g. The three dimensional form of the tablets is arbitrary andmay circular, oval or angular, with intermediate forms also beingpossible.

At least one of the phases in multi-phase agents, that contains anamylase-sensitive material, in particular starch, or is at leastpartially surrounded or coated by said material, is particularlyadvantageous. Said phase is mechanically stabilized and/or protectedfrom influences from the outside in this way and is, at the same time,attacked via an amylase active in the wash liquor, so as to facilitaterelease of the ingredients.

Agents according to the invention that are likewise preferred arecharacterized in that they are overall in a liquid, gel-like orpaste-like form. The proteins contained therein, preferably a protein ofthe invention, are added to such agents, preferably starting fromprotein isolation and preparation carried out according to the priorart, in a concentrated aqueous or nonaqueous solution, for example inliquid form, for example as solution, suspension or emulsion, but alsoin gel form or encapsulated or as dried powder. Such detergents orcleansers according to the invention in the form of solutions incustomary solvents are usually prepared by simply mixing the ingredientswhich can be introduced as solids or as a solution into an automatedmixer.

One embodiment of the present invention are those agents in liquid, gelor paste form, to which a protein essential to the invention and/or anyof the other contained proteins and/or any of the other containedingredients has been added in encapsulated form, preferably in the formof microcapsules. Among these, particular preference is given to thosewith capsules made of amylase-sensitive material. Such a combined use ofamylase-sensitive materials and the amylolytic enzyme essential to theinvention in a detergent or cleanser may exhibit synergistic effects,for example such that the starch-cleaving enzyme assists cleavage of themicrocapsules and thus controls the process of releasing theencapsulated ingredients so that the latter are released not duringstorage and/or not at the beginning of the purification process but onlyat a particular time. This mechanism may be the basis of complexdetergent and cleanser systems with a large variety of ingredients andcapsule types, which are particularly preferred embodiments of thepresent invention.

A comparable effect arises when the ingredients of the detergent orcleanser are distributed over at least two different phases, for exampletwo or more solid phases, connected to one another, of a tablet-likedetergent or cleanser, or different granules within the same pulverulentagent. Two- or multiphase cleaners are state of the art for applicationboth in machine dishwashers and in detergents. The activity of anamylolytic enzyme in a previously activated phase is a precondition foractivation of a later phase, if the latter is surrounded by anamylase-sensitive envelope or coating or if the amylase-sensitivematerial is an integral component of the solid phase, which, whenpartially or completely hydrolyzed, leads to disintegration of the phasein question. The use of the enzyme essential to the invention for thispurpose is thus a preferred embodiment of the present invention.

The ingredients of detergents and cleansers are suitably able to supporteach other's performance. The application WO 99/63035, for example,discloses the synergistic use of amylase and color transfer inhibitorsin order to increase cleaning performance. It has also been disclosed,for example in the application WO 98/45396, that polymers which may beused simultaneously as cobuilders, such as, for example, alkylpolyglycosides, can stabilize and increase the activity and stability ofenzymes present. Preference is therefore given to an α-amylase variantaccording to the invention being modified, in particular stabilized,and/or its contribution to the washing or cleaning performance of theagent being increased by any of the other components mentioned above.Appropriately adjusted formulations for agents according to theinvention are thus particularly preferred embodiments of the presentinvention.

Within appropriate agents, α-amylase variants according to the inventionmay serve to activate their own or other phases, if they are providedalone or together with at least one other cleaning-active or cleaningaction-supporting substance in a detergent or cleanser consisting ofmore than one phase. Accordingly, they may also serve to releaseingredients from capsules, if they or another active substance areprovided in encapsulated form in a detergent or cleanser.

The invention also relates to processes for cleaning textiles or hardsurfaces, which are characterized in that an above-described α-amylasevariant according to the invention becomes active in at least one of theprocess steps.

This is because this embodiment implements the invention in that theimproved enzymic properties according to the invention, in particularthe increased stability to multimerization, are beneficial in principleto any cleaning process, for example in that less enzyme is lost due toaggregation, even during application. Each cleaning process is enrichedby the activity in question, if the latter is added in at least oneprocess step. Processes of this kind are carried out, for example, usingmachines such as familiar domestic dishwashers or domestic washingmachines. Preference is accordingly given to preferred processesaccording to the above statements.

Further preference is given to those methods which are characterized inthat the α-amylase variant is used by way of an above-described agentaccording to the invention.

Particular preference is given to any method characterized in that theα-amylase is employed in the step in question in an amount of from 0.01mg to 400 mg per corresponding step, preferably from 0.02 mg to 300 mg,particularly preferably from 0.03 mg to 100 mg.

Advantageously, this results in concentrations of from 0.0005 to 20 mgper l, preferably 0.005 to 10 mg per l, particularly preferably 0.005 to8 mg of the amylolytic protein per l of wash liquor. The proteinconcentration may be determined with the aid of known methods, forexample the abovementioned BCA or biuret methods.

According to the above specification, the present invention is alsoimplemented by the use of α-amylase variants of the invention becausehere too, the advantageous properties of the enzymes in question areeffective. This applies in particular to cleaning purposes.

A separate subject matter of the invention is therefore the use of anyof the above-described α-amylase variants according to the invention forcleaning textiles or hard surfaces.

It is possible here to use said variant as the sole active component,preferentially together with at least other cleaning-active or cleaningaction-supporting substance.

Preference is therefore given to such a use that is characterized inthat the α-amylase variant is used via an above-described agentaccording to the invention.

Advantageously, such a use is characterized in that from 0.01 mg to 400mg of the α-amylase variant, preferably from 0.02 mg to 300 mg,particularly preferably from 0.03 mg to 100 mg, are employed perapplication, preferably per application in a dishwasher or a washingmachine.

This is because this produces advantageously the concentrations listedabove in the wash liquor. This metering may be carried out by themanufacturer of said agent or by the end user, depending on the cleaningproblem.

Another embodiment is the use of an α-amylase variant according to theinvention for the treatment of raw materials or intermediate products intextile manufacture, in particular for desizing cotton.

Raw materials and intermediates in the manufacture of textiles, forexample those based on cotton, are provided with starch during theirproduction and further processing, in order to improve processing. Thismethod which is applied to yarns, to intermediates and to textiles iscalled sizing. Amylolytic proteins according to the invention aresuitable for removing the starch-containing protective layer (desizing),in particular because they are stabilized to multimerization during theaction in a liquid medium.

The following examples further illustrate the present invention.

EXAMPLES

All molecular-biological steps are carried out following standardmethods as described, for example, in the manual by Fritsch, Sambrookand Maniatis “Molecular cloning: a laboratory manual”, Cold SpringHarbour Laboratory Press, New York, 1989, or comparable specialistliterature. Enzymes, kits and apparatus are used according to theinstructions by the respective manufacturers.

Example 1

Culturing of Bacillus sp. A 7-7 (DSM 12368)

The microorganism Bacillus sp. A 7-7 has been deposited under thedeposit number DSM 12368 with the Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig, Germany(http://www.dsmz.de). It is described in the application WO 02/10356 A2.The DNA and amino acid sequences of the α-amylase produced by this(deposited) species differ from the sequences depicted in the sequencelisting of WO 02/10356 A2 in the following two positions: thecorresponding DNA has, in nucleic acid positions 805-807 according toSEQ ID NO. 1 of the present application, the triplet gat which codes forthe amino acid D (in amino acid position 236), and, in positions1156-1158, the triplet tat which codes for the amino acid Y (in position353). (SEQ ID NO. 1 and 2 of WO 02/10356 A2 indicate the triplets ggtfor G and tgt for C, respectively, at the corresponding positions.)

Using well-known methods of point mutagenesis, for example with the aidof the QuickChange kit from Stratagene®, La Jolla, USA (see below), andon the basis of primers derived from SEQ ID NO. 1, it is possible toconvert this α-amylase to a different one.

WO 02/10356 A2 describes culturing of Bacillus sp. A 7-7 (DSM 12368). Asuitable medium is YPSS medium containing 15 g/l soluble starch, 4 g/lyeast extract, 1 g/l K₂HPO₄ and 0.5 g/l MgSO₄×7H₂O, with the pH beingadjusted to 10.3 with 20% strength sodium carbonate solution afterautoclaving. From this the α-amylase in question can be obtained, aslikewise illustrated in WO 02/10356 A2. Thus it is possible for saidα-amylase and the variants derived therefrom to be prepared bywell-known methods, at least on the laboratory scale.

Example 2

Homology Modeling and Selection of the Amino Acids Replaceable Accordingto the Invention

Homology Modeling

Homology modeling for Bacillus sp. A 7-7 (DSM 12368) α-amylase wascarried out via the RSCB protein database (accessible viaMax-Delbrück-Zentrum in Berlin, Germany), as illustrated in thedescription. In this connection, the search using the protein sequencedetected the following structures: B. licheniformis α-amylase (RCSB-PDBdatabase entry: 1 BLI), a chimeric α-amylase of those of B.amyloliquefaciens and B. licheniformis, in its native structure (1E3X,1E43), an acarbose complex of the same chimeric α-amylase (1E3Z), aTris/maltotriose complex of the same chimeric α-amylase (1E40) and akinetically stabilized variant of B. licheniformis α-amylase (10B0).

By superimposing these structure in the SwissPDB viewer, a leaderstructure was generated onto which the protein sequence of ALBA was thenmodeled. The orientation of the side chains in the ALBA structure wasthen corrected and energy minimization was carried out. The individualsteps can be found in the user manual mentioned and were carried outaccording to the standard settings of the program.

Overall, the following 407 amino acids are located on the surface of themolecule which comprises 484 amino acids (they are defined as such byway of an accessibility of at least 1; regarding accessibility: seedescription and subsequent section):

-   -   T5, N6, G7, T8, M9, Q11, Y12, E14, W15, Y16, L17, P18, N19, D20,        G21, N22, H23, W24, N25, R26, R28, S29, D30, A31, S32; N33, K35,        D36, K37, G38, I39, T40, A41, W43, P46, A47, W48, K49, G50, A51,        S52, Q53, N54, D55, V56, G57, Y58, G59, A60, Y61, D62 , L63,        Y64, L66, G67, E68, F69, N70, Q71, K72, G73, T74, V75, R76, T77,        K78, Y79, G80, T81, R82, N83, Q84, L85, Q86, A87, V89, T90, A91,        K93, S94, N95, G96, Q98, V99, Y100, V103, M105 N106, H107, K108,        G110, A111, D112, A113 T114, E115, W116, V117, R118, V120, E121,        V122, N123, P124, S125, N126, R127, N128, Q129, E130, V131,        S132, G133, D134, Y135, T136, I137, E138, W140, K142, F143,        D144, F145, P146, G147, R148, G149, N150, T151, H152, S153,        N154, F155, K156, W157, R158, W159, Y160, H161, D166, W167,        D168, Q169, S170, R171, Q172, L173, Q174, N175, R176, I177,        Y178, K179, R181, G182, D183, G184, K185, G186, W187, W189,        E190, V191, D192, T193, E194, N195, G196, N197, Y198, D199,        Y200, L201, M202, Y203, I206, D207, M208, D209, H210, P211,        E212, V214, N215, E216, L217, R218, N219, V222, W223, T225,        N226, T227, L228, G229, L230, D231, F233, R234, I235, G236,        A237, K239, H240, I241, K242, Y243, S244, F245, T246, R247,        G248, W249, L250, T251, H252, V253, R254, N255, T256, T257,        G258, K259, N260, M261, F262, A263, E266, F267, W268, K269,        N270, D271, I272 G273, A274, I275, E276, N277, S280, K281, N283,        W284, N285, H286, S287, V288, F289, P292, L293, Y295, N296,        L297, Y298, N299, S301, R302, S303, G304, G305, N306, Y307,        D308, M309, R310, Q311, I312, F313, N314, G315, V318, Q319,        R320, H321, P322, T323, H324, T327, F328, V329, D330, N331,        H332, D333, Q335, P336, E337, E338, A339, L340, E341, S342,        F343, E345, E346, W347, F348, K349, P350, L351, C353, L355,        T356, L357, R359, D360, Q361, G362, Y363, S365, V366, F367,        Y368, D370, Y371, Y372, G373, I374, P375, T376, H377, G378,        P380, A381, M382, K383, S384, K385, I386, D387, P388, L390,        E391, R393, Q394, K395, Y396, Y398, G399, K400, Q401, N402,        D403, Y404, L405, D406, H407, H408, N409, M410, R415, E416,        G417, N418, T419, A420, H421, P422, N423, S424, M430, D432,        G433, P434, G435, G436, N437, K438, W439, Y441, G443, R444,        N445, K446, A447, G448, Q449, V450, W451, R452, D453, I454,        T455, G456, N457, R458, S459, G460, T461, V462, T463, I464,        N465, A466, D467, W469, N471, S473, V474, N475, G476, G477,        S478, V479, V483, N484, N485

This includes also the following 44 asparagine residues:

-   -   N6, N19, N22, N25, N33, N54, N70, N83, N95, N106, N123, N126,        N128, N150, N154, N175, N195, N197, N215, N219, N226, N255,        N260, N270, N277, N283, N285, N296, N299, N306, N314, N331,        N402, N409, N418, N423, N437, N445, N457, N465, N471, N475,        N484, N485.

This includes further the following 17 glutamine residues:

-   -   Q11, Q53, Q71, Q84, Q86, Q98, Q129, Q169, Q172, Q174, Q311,        Q319, Q335, Q361, Q394, Q401, Q449.

These asparagine and glutamine residues are emphasized by color in theimage of Bacillus sp. A 7-7 (DSM 12368) α-amylase in FIGS. 1 and 2.

Calculation of Solvent Accessibility

Based on these results, the solvent accessibility of the above-describedamino acid residues N and Q located on the surface were then calculated.To this end, the above-mentioned SwissPdb viewer was used again,preserving the standard parameters of the program. As a result, thefollowing solvent accessibility values were determined for the aminoacid residues found and are indicated in each case in % in bracketsafter the positions listed:

-   -   N6 (12), N19 (28), N22 (28), N25 (16), N33 (28), N54 (35), N70        (38), N83 (44), N95 (19), N106 (1), N123 (19), N126 (18), N128        (31), N150 (39), N154 (41), N175 (20), N195 (24), N197 (3), N215        (27), N219 (32), N226 (40), N255 (42), N260 (49), N270 (38),        N277 (24), N283 (40), N285 (27), N296 (15), N299 (23), N306        (51), N314 (35), N331 (6), N402 (16), N409 (11), N418 (25), N423        (40), N437 (37), N445 (41), N457 (29), N465 (22), N471 (20),        N475 (25), N484 (12), N485 (38);        and:    -   Q11 (4), Q53 (12), Q71 (5), Q84 (24), Q86 (18), Q98 (29), Q129        (31), Q169 (26), Q172 (53), Q174 (44), Q311 (35), Q319 (38),        Q335 (3), Q361 (36), Q394 (20), Q401 (14), Q449 (31).

Of these, the following 41 and 14 have thus an accessibility of at least10%:

-   -   N6 (12), N19 (28), N22 (28), N25 (16), N33 (28), N54 (35), N70        (38), N83 (44), N95 (19), N123 (19), N126 (18), N128 (31), N150        (39), N154 (41), N175 (20), N195 (24), N215 (27), N219 (32),        N226 (40), N255 (42), N260 (49), N270 (38), N277 (24), N283        (40), N285 (27), N296 (15), N299 (23), N306 (51), N314 (35),        N402 (16), N409 (11), N418 (25), N423 (40), N437 (37), N445        (41), N457 (29), N465 (22), N471 (20), N475 (25), N484 (12),        N485 (38);        and, respectively:    -   Q53 (12), Q84 (24), Q86 (18), Q98 (29), Q129 (31), Q169 (26),        Q172 (53), Q174 (44), Q311 (35), Q319 (38), Q361 (36), Q394        (20), Q401 (14), Q449 (31).

Of these, the following 32 and 11 have thus an accessibility of at least20%:

-   -   N19 (28), N22 (28), N33 (28), N54 (35), N70 (38), N83 (44), N128        (31), N150 (39), N154 (41), N175 (20), N195 (24), N215 (27),        N219 (32), N226 (40), N255 (42), N260 (49), N270 (38), N277        (24), N283 (40), N285 (27), N299 (23), N306 (51), N314 (35),        N418 (25), N423 (40), N437 (37), N445 (41), N457 (29), N465        (22), N471 (20), N475 (25), N485 (38);        and, respectively:    -   Q84 (24), Q98 (29), Q129 (31), Q169 (26), Q172 (53), Q174 (44),        Q311 (35), Q319 (38), Q361 (36), Q394 (20), Q449 (31).

Of these, the following 18 and 7 thus have an accessibility of at least30%:

-   -   N54 (35), N70 (38), N83 (44), N128 (31), N150 (39), N154 (41),        N219 (32), N226 (40), N255 (42), N260 (49), N270 (38), N283        (40), N306 (51), N314 (35), N423 (40), N437 (37), N445 (41),        N485 (38);        and, respectively:    -   Q129 (31), Q172 (53), Q174 (44), Q311 (35), Q319 (38), Q361        (36), Q449 (31).

Example 3

Site-Specific Mutagenesis

For Bacillus sp. A 7-7 (DSM 12368) α-amylase, the positions determinedin the previous example serve as starting points for point mutations viasite-directed mutagenesis, i.e. for introducing a different amino acidto the position(s) in question. Said mutagenesis is carried out, forexample, with the aid of the QuikChange kit (Stratagene, cat. No.200518) according to the corresponding protocol. The primers may bedesigned on the basis of the DNA and amino acid sequences indicated inSEQ ID NO. 1, the particular codon being altered according to the aminoacid to be introduced.

According to this principle, an expression vector containing theα-amylase sequence is suitably mutagenized accordingly and transformedinto an expression strain in the present example into B. subtilis, bywell-known methods.

Example 4

Production and Purification of Amylase Mutants

Amylase-positive B. subtilis strains are grown in the YPSS mediummentioned in Example 1. This procedure and purification of the enzymeproduced by these strains are carried out according to the descriptionin WO 02/10356 A2. The latter also reveals determination of theamylolytic activity of the purified enzyme according to the “DNSmethod”. The activity determinable in this way serves hereinbelow asparameter for the stability of the enzyme under in each case differentconditions.

Determination of Solvent Stability

The solvent stability of the variants in comparison with the respectivestarting enzymes, i.e. the wild type enzymes or those which specificallydo not have the substitutions of the invention, is suitably measured ina buffer of pH 11 at 40° C., with 30 minutes of incubation. This isfollowed by the activity assay described in the application WO 02/10356A2, in which assay variants according to the invention producesignificantly superior values.

Abbreviations: A 7-7: α-amylase of Bacillus sp. A 7-7 (DSM 12368; SEQ IDNO. 2) S707: α-amylase of Bacillus sp. #707 LAMY: α-amylase of Bacillussp. KSM-AP1378 BAA: α-amylase of B. amyloliquefaciens BLA: α-amylase ofB. licheniformis BStA: α-amylase of B. stearothermophilus MK716:α-amylase of Bacillus sp. MK716 TS-23: α-amylase of Bacillus sp. TS-23K38: α-amylase of Bacillus KSM-K38

1. An α-amylase variant having at least one amino acid substitution withrespect to a predetermined α-amylase starting molecule, wherein in saidat least one amino acid substitution an asparagine (N) or a glutamine(Q) residue on the surface of said starting molecule is replaced with A,C, F, G, H, I, K, L, M, P, R, S, T, V, W or Y in the variant.
 2. Anα-amylase variant according to claim 1, wherein in said amino acidsubstitution N is replaced with A, G, K, R, S or T, or Q is replacedwith A, G, I, K, R, S or T.
 3. An α-amylase variant according to claim1, wherein said asparagine (N) glutamine (Q) residue has anaccessibility of at least 10% prior to amino acid substitution, saidaccessibility of the residue being calculated on a scale from 0% (notaccessible to the solvent) to 100% (present in a hypotheticalpentapeptide, GGXGG).
 4. An α-amylase variant according to claim 1having from 2 to 10 amino acid substitutions.
 5. An α-amylase variantaccording to 1, wherein the starting molecule is selected from the groupconsisting of α-amylase from Bacillus sp. A 7-7 (DSM 12368), α-amylasefrom Bacillus sp. #707, a amylase from Bacillus sp. KSM-AP1378,α-amylase from Bacillus KSM-K38, α-amylase from B. amyloliquefaciens,α-amylase from B. licheniformis, α-amylase from Bacillus sp. MK716,α-amylase from Bacillus sp. TS-23, α-amylase from B. stearothermophilus,α-amylase from B. agaradherens, and cyclodextrin glucanotransferase(CGTase) from B. agaradherens; hybrid amylases therefrom; and α-amylasesderived therefrom by mutagenesis of at least one amino acid.
 6. Anα-amylase variant according to claim 5, wherein the starting molecule isselected from the group consisting of α-amylase from Bacillus sp. A 7-7(DSM 12368), cyclodextrin glucanotransferase from B. agaradherens (DSM9948), and hybrid amylases of the α-amylases from B. amyloliquefaciensand from B. licheniformis.
 7. An α-amylase variant according to claim 6,wherein the starting molecule is selected from the group consisting ofthe hybrid amylases AL34, AL76, AL112, AL256, ALA34-84, LAL19-153, andLAL19-433.
 8. An α-amylase variant according to claim 1, wherein thestarting molecule is an α-amylase whose amino acid sequence is at least96% identical to the amino acid sequence indicated in SEQ ID NO. 2 inpositions +1 to
 484. 9. An α-amylase variant according to claim 1,wherein the starting molecule is an α-amylase variant containing atleast one additional point mutation.
 10. An α-amylase variant accordingto claim 9, wherein the at least one additional point mutation isselected from the group consisting of the positions: 5, 167, 170, 177,202, 204, 271, 330, 377, 385 and 445, according to the numbering of themature protein according to SEQ ID NO.
 2. 11. An α-amylase variantaccording to claim 10, wherein the at least one additional pointmutation is selected from the group consisting of 5A, 167R, 170P, 177L,202L, 204V, 271 D, 330D, 377R, 385S and 445Q.
 12. A method of preparinga variant α-amylase having increased stability with respect tosolvent-exerted hydrolysis in comparison with a predetermined α-amylasestarting molecule, the method comprising: (a) identifying at least oneasparagine (N) or glutamine (Q) amino acid residue on the surface of thepredetermined α-amylase starting molecule, and (b) preparing a variantα-amylase whereby the at least one asparagine (N) or glutamine (Q)residue on the surface of said predetermined α-amylase molecule isreplaced with A, C, F, G, H, I, K, L, M, P, R, S, T, V, W or Y.
 13. Amethod according to claim 12, wherein the increased stability of thevariant a amylase to solvent-exerted hydrolysis is exhibited at elevatedtemperatures or high pH.
 14. A method according to claim 12, wherein Nis replaced with A, G, K, R, S or T, or Q is replaced with A, G, I, K,R, S or T.
 15. A method according to claim 12, wherein said at least oneamino acid residue has an accessibility of at least 10% prior to aminoacid substitution, said accessibility of the amino acid residue beingcalculated on a scale from 0% (not accessible to the solvent) to 100%(present in a hypothetical pentapeptide, GGXGG).
 16. A method accordingto claim 11, wherein the predetermined α-amylase is selected from thegroup consisting of α-amylase from Bacillus sp. A 7-7 (DSM 12368),α-amylase from Bacillus sp. #707, a amylase from Bacillus sp.KSM-AP1378, α-amylase from Bacillus KSM-K38, α-amylase from B.amyloliquefaciens, α-amylase from B. licheniformis, α-amylase fromBacillus sp. MK716, α-amylase from Bacillus sp. TS-23, α-amylase from B.stearothermophilus, α-amylase from B. agaradherens, and cyclodextringlucanotransferases (CGTase) from B. agaradherens, hybrid amylasestherefrom, and α-amylases derived therefrom by mutagenesis of at leastone amino acid.
 17. A method according to claim 16, wherein thepredetermined α-amylase is selected from the group consisting ofα-amylase from Bacillus sp. A 7-7 (DSM 12368), cyclodextringlucanotransferase from B. agaradherens (DSM 9948) and a hybrid amylaseof the α-amylases from B. amyloliquefaciens and from B. licheniformis.18. A method according to claim 12, the predetermined α-amylase startingmolecule being an α-amylase whose amino acid sequence is at least 96%identical to the amino acid sequence indicated in SEQ ID NO. 2 inpositions +1 to
 485. 19. A method according to claim 12, wherein thepredetermined α-amylase starting molecule is an α-amylase variantcontaining at least one additional point mutation.
 20. A nucleic acidcoding for an α-amylase variant according to claim
 1. 21. A nucleic acidaccording to claim 20, the nucleic acid being derived from a nucleicacid according to SEQ ID NO. 1 or from a variant thereof having at leastone additional point mutation to give those mutations resulting in an atleast one amino acid substitution wherein, in said at least one aminoacid substitution, an asparagine (N) or a glutamine (Q) residue on thesurface of said starting molecule is replaced with A, C, F, G, H, I, K,L, M, P, R, S, T, V, W or Y in the variant.
 22. A vector comprising anucleic acid according to claim
 20. 23. A cloning vector according toclaim
 22. 24. An expression vector according to claim
 22. 25. A cellcomprising, after genetic modification, a nucleic acid according toclaim
 20. 26. A cell according to claim 25, said nucleic acid being partof a vector.
 27. A cell according to claim 25, the cell being abacterium that secretes the α-amylase variant formed into thesurrounding medium.
 28. A cell according to claim 25, the cell being aGram-negative bacterium selected from the group consisting of thespecies Escherichia coli, the genus Klebsiella, the genus Pseudomonas,and the genus Xanthomonas.
 29. A cell according to claim 28, the cellbeing selected from the group consisting of the strains Escherichia coliK12 and E. coli B, and of derivatives of the strains Escherichia coliBL21 (DE3), E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 andKlebsiella planticola (Rf).
 30. A cell according to claim 25, the cellbeing a Gram-positive bacterium selected from the group consisting ofthe genera Bacillus, Staphylococcus and Corynebacterium.
 31. A cellaccording to claim 30, the cell being selected from the group consistingof the species Bacillus lentus, B. licheniformis, B. amyloliquefaciens,B. subtilis, B. globigii and B. alcalophilus, Staphylococcus carnosusand Corynebacterium glutamicum.
 32. A detergent or cleaning agentcomprising an α-amylase variant according to claim
 1. 33. An agentaccording to claim 32, comprising from 0.000001 percent by weight to 5%by weight of the α-amylase variant.
 34. An agent according to claim 32,further including at least one additional enzyme selected from the groupconsisting of amylases, proteases, lipases, cutinases, hemicellulases,cellulases, β-glucanases, oxidases, peroxidases, perhydrolases andlaccases.
 35. An agent according to claim 32 further comprising at leastone additional component, wherein the α-amylase variant is stabilized,and/or its contribution to the washing or cleaning performance of theagent is increased, by the at least one additional component.
 36. Anagent according to claim 32, the agent being a solid including at leastone compacted component.
 37. An agent according to claim 32, the agentbeing liquid, gel-like or paste-like, the α-amylase variant beingencapsulated.
 38. A method of cleaning textiles or hard surfaces, themethod comprising: (a) applying an agent including at least oneα-amylase variant according to claim 1 to a textile of hard surfacesubstrate, and (b) activating the at least one α-amylase variant.
 39. Amethod according to claim 38, wherein the agent comprises from 0.01 mgto 400 mg of the at least one α-amylase variant.
 40. A cell according toclaim 30, the cell being a derivative of B. licheniformis DSM 13.